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Sample records for a-induced cell damage

  1. Withaferin A Induces Oxidative Stress-Mediated Apoptosis and DNA Damage in Oral Cancer Cells

    PubMed Central

    Chang, Hsueh-Wei; Li, Ruei-Nian; Wang, Hui-Ru; Liu, Jing-Ru; Tang, Jen-Yang; Huang, Hurng-Wern; Chan, Yu-Hsuan; Yen, Ching-Yu

    2017-01-01

    Withaferin A (WFA) is one of the most active steroidal lactones with reactive oxygen species (ROS) modulating effects against several types of cancer. ROS regulation involves selective killing. However, the anticancer and selective killing effects of WFA against oral cancer cells remain unclear. We evaluated whether the killing ability of WFA is selective, and we explored its mechanism against oral cancer cells. An MTS tetrazolium cell proliferation assay confirmed that WFA selectively killed two oral cancer cells (Ca9-22 and CAL 27) rather than normal oral cells (HGF-1). WFA also induced apoptosis of Ca9-22 cells, which was measured by flow cytometry for subG1 percentage, annexin V expression, and pan-caspase activity, as well as western blotting for caspases 1, 8, and 9 activations. Flow cytometry analysis shows that WFA-treated Ca9-22 oral cancer cells induced G2/M cell cycle arrest, ROS production, mitochondrial membrane depolarization, and phosphorylated histone H2A.X (γH2AX)-based DNA damage. Moreover, pretreating Ca9-22 cells with N-acetylcysteine (NAC) rescued WFA-induced selective killing, apoptosis, G2/M arrest, oxidative stress, and DNA damage. We conclude that WFA induced oxidative stress-mediated selective killing of oral cancer cells. PMID:28936177

  2. Cyclosporine A-induced apoptosis in renal tubular cells is related to oxidative damage and mitochondrial fission.

    PubMed

    de Arriba, Gabriel; Calvino, Miryam; Benito, Selma; Parra, Trinidad

    2013-03-27

    Cyclosporine A (CsA) nephrotoxicity has been linked to reactive oxygen species (ROS) production in renal cells. We have demonstrated that the antioxidant Vitamin E (Vit E) abolished renal toxicity in vivo and in vitro models. As one of the main sources of intracellular ROS are mitochondria, we studied the effects of CsA on several mitochondrial functions in LLC-PK1 cells. CsA induced ROS synthesis and decreased reduced glutathione (GSH). The drug decreased mitochondrial membrane potential (ΔΨm) and induced physiological modifications in both the inner (IMM) and the outer mitochondrial membranes (OMM). In the IMM, CsA provoked mitochondrial permeability transition pores (MPTP) and cytochrome c was liberated into the intermembrane space. CsA also induced pore formation in the OMM, allowing that intermembrane space contents can reach cytosol. Furthermore, CsA altered the mitochondrial dynamics, inducing an increase in mitochondrial fission; CsA increased the expression of dynamin related protein 1 (Drp1) that contributes to mitochondrial fission, and decreased the expression of mitofusin 2 (Mfn2) and optic atrophy protein 1 (Opa1), proteins involved in the fusion process. All these phenomena were related to apoptosis. These effects were inhibited when cells were treated with the antioxidant Vit E suggesting that they were mediated by the synthesis of ROS. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  3. Catechins: natural free-radical scavengers against ochratoxin A-induced cell damage in a pig kidney cell line (LLC-PK1).

    PubMed

    Costa, S; Utan, A; Cervellati, R; Speroni, E; Guerra, M C

    2007-10-01

    Besides aflatoxin B1, recent findings suggested that oxidative stress plays an important role in the toxicity of an other mycotoxin: ochratoxin A (OTA). The protective effect of two catechins (epigallocatechin gallate, EGCG, and epicatechin gallate, ECG) against OTA-induced cytotoxicity was investigated in a pig kidney cell line (LLC-PK1). The ability of the catechins to reduce ROS production and DNA fragmentation induced by OTA was also investigated. Our experiments proved the significant cytoprotective effects of the molecules in vitro from OTA-induced cell damage. In particular a 24h pre-treatment with EGCG or ECG restored cell viability with respect to OTA alone. Pre-treatment with EGCG at low concentration for 8 days protected cells from OTA-induced cell death. Moreover both catechins reduced OTA-induced ROS production. A reduction of OTA-induced DNA fragmentation was found for LLC-PK1 cells pre-treated with EGCG and ECG. The free-radical scavenging capacity of both catechins was tested with the Briggs-Rauscher oscillating method (pH approximately 2) and the TEAC assay (pH 7.4). The results show a good scavenging power according with inhibition of ROS production. Catechins could be useful to develop alimentary strategies for both humans and animals to prevent OTA-induced cytotoxicity.

  4. The American Cockroach Peptide Periplanetasin-2 BlocksClostridium DifficileToxin A-Induced Cell Damage and Inflammation in the Gut.

    PubMed

    Hong, Ji; Zhang, Peng; Yoon, I Na; Hwang, Jae Sam; Kang, Jin Ku; Kim, Ho

    2017-04-28

    Clostridium difficile , which causes pseudomembranous colitis, releases toxin A and toxin B. These toxins are considered to be the main causative agents for the disease pathogenesis, and their expression is associated with a marked increase of apoptosis in mucosal epithelial cells. Colonic epithelial cells are believed to form a physical barrier between the lumen and the submucosa, and abnormally increased mucosal epithelial cell apoptosis is considered to be an initial step in gut inflammation responses. Therefore, one approach to treating pseudomembranous colitis would be to develop agents that block the mucosal epithelial cell apoptosis caused by toxin A, thus restoring barrier function and curing inflammatory responses in the gut. We recently isolated an antimicrobial peptide, Periplanetasin-2 (Peri-2, YPCKLNLKLGKVPFH) from the American cockroach, whose extracts have shown great potential for clinical use. Here, we assessed whether Peri-2 could inhibit the cell toxicity and inflammation caused by C. difficile toxin A. Indeed, in human colonocyte HT29 cells, Peri-2 inhibited the toxin A-induced decrease in cell proliferation and ameliorated the cell apoptosis induced by this toxin. Moreover, in the toxin A-induced mouse enteritis model, Peri-2 blocked the mucosal disruption and inflammatory response caused by toxin A. These results suggest that the American cockroach peptide Peri-2 could be a possible drug candidate for addressing the pseudomembranous colitis caused by C. difficile toxin A.

  5. Ectopic expression of H2AX protein promotes TrkA-induced cell death via modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage

    SciTech Connect

    Jung, Eun Joo; Kim, Deok Ryong, E-mail: drkim@gnu.ac.kr

    2011-01-21

    Research highlights: {yields} We established TrkA-inducible U2OS cells stably expressing GFP-H2AX proteins. {yields} GFP-H2AX was colocalized with TrkA in the cytoplasm. {yields} {gamma}H2AX production was significantly increased upon activation of TrkA and suppressed by TrkA inhibitor or JNK inhibitor. {yields} Ectopic expression of H2AX promoted TrkA-mediated cell death through the modulation of TrkA tyrosine-490 phosphorylation and JNK activity upon DNA damage. -- Abstract: We previously reported that TrkA overexpression causes accumulation of {gamma}H2AX proteins in the cytoplasm, subsequently leading to massive cell death in U2OS cells. To further investigate how cytoplasmic H2AX is associated with TrkA-induced cell death, we establishedmore » TrkA-inducible cells stably expressing GFP-tagged H2AX. We found that TrkA co-localizes with ectopically expressed GFP-H2AX proteins in the cytoplasm, especially at the juxta-nuclear membranes, which supports our previous results about a functional connection between TrkA and {gamma}H2AX in TrkA-induced cell death. {gamma}H2AX production from GFP-H2AX proteins was significantly increased when TrkA was overexpressed. Moreover, ectopic expression of H2AX activated TrkA-mediated signal pathways via up-regulation of TrkA tyrosine-490 phosphorylation. In addition, suppression of TrkA tyrosine-490 phosphorylation under a certain condition was removed by ectopic expression of H2AX, indicating a functional role of H2AX in the maintenance of TrkA activity. Indeed, TrkA-induced cell death was highly elevated by ectopic H2AX expression, and it was further accelerated by DNA damage via JNK activation. These all results suggest that cytoplasmic H2AX could play an important role in TrkA-mediated cell death by modulating TrkA upon DNA damage.« less

  6. DNA Damage in Stem Cells.

    PubMed

    Vitale, Ilio; Manic, Gwenola; De Maria, Ruggero; Kroemer, Guido; Galluzzi, Lorenzo

    2017-05-04

    Both embryonic and adult stem cells are endowed with a superior capacity to prevent the accumulation of genetic lesions, repair them, or avoid their propagation to daughter cells, which would be particularly detrimental to the whole organism. Inducible pluripotent stem cells also display a robust DNA damage response, but the stability of their genome is often conditioned by the mutational history of the cell population of origin, which constitutes an obstacle to clinical applications. Cancer stem cells are particularly tolerant to DNA damage and fail to undergo senescence or regulated cell death upon accumulation of genetic lesions. Such a resistance contributes to the genetic drift of evolving tumors as well as to their limited sensitivity to chemo- and radiotherapy. Here, we discuss the pathophysiological and therapeutic implications of the molecular pathways through which stem cells cope with DNA damage. Copyright © 2017 Elsevier Inc. All rights reserved.

  7. Gallium arsenide solar cell radiation damage study

    NASA Technical Reports Server (NTRS)

    Maurer, R. H.; Herbert, G. A.; Kinnison, J. D.; Meulenberg, A.

    1989-01-01

    A thorough analysis has been made of electron- and proton- damaged GaAs solar cells suitable for use in space. It is found that, although some electrical parametric data and spectral response data are quite similar, the type of damage due to the two types of radiation is different. An I-V analysis model shows that electrons damage the bulk of the cell and its currents relatively more, while protons damage the junction of the cell and its voltages more. It is suggested that multiple defects due to protons in a strong field region such as a p/n junction cause the greater degradation in cell voltage, whereas the individual point defects in the quasi-neutral minority-carrier-diffusion regions due to electrons cause the greater degradation in cell current and spectral response.

  8. Radiation damage of gallium arsenide production cells

    NASA Technical Reports Server (NTRS)

    Mardesich, N.; Garlick, G. F. J.

    1987-01-01

    High-efficiency gallium arsenide cells, made by the liquid epitaxy method (LPE), have been irradiated with 1-MeV electrons up to fluences of 10 to the 16th e/sq cm. Measurements have been made of cell spectral response and dark and light-excited current-voltage characteristics and analyzed using computer-based models to determine underlying parameters such as damage coefficients. It is possible to use spectral response to sort out damage effects in the different cell component layers. Damage coefficients are similar to other reported in the literature for the emitter and buffer (base). However, there is also a damage effect in the window layer and possibly at the window emitter interface similar to that found for proton-irradiated liquid-phase epitaxy-grown cells. Depletion layer recombination is found to be less than theoretically expected at high fluence.

  9. Paeoniflorin exerts a nephroprotective effect on concanavalin A-induced damage through inhibition of macrophage infiltration.

    PubMed

    Liu, Cheng; Cheng, Zhuoan; Wang, Yunman; Dai, Xiuqin; Zhang, Jie; Xue, Dongying

    2015-07-25

    It is well established that macrophage infiltration is involved in concanavalin A (conA)-induced liver injury. However, the role of macrophages in conA-induced renal injury remains unknown. The aims of this study were to investigate macrophage infiltration in conA-induced renal injury and determine whether paeoniflorin (PF) could inhibit macrophage infiltration into the kidney. BALB/C mice were pre-treated with or without PF 2 h (h) before conA injection. At 8 h after con A injection, all the mice were sacrificed; The liver and kidney histology were studied. The renal CD68 expression was detected by immunohistochemical and real-time PCR analysis. The level of expression of C-X-C chemokine receptor type 3 (CXCR3) was analyzed by western blot, immunohistochemical and real-time PCR. The pathophysiological involvement of CXCR3 in macrophage infiltration were investigated using dual-colour immunofluorescence microscopy. PF administration significantly reduced the elevated serum levels of alanine transaminase (ALT), blood urea nitrogen (BUN), creatinine (Cr) and the severity of liver and renal damage compared with that in the conA-vehicle group. PF administration inhibited the increase in renal IL1β mRNA expression and concentration. Furthermore, immunohistochemical analysis showed that macrophages secreted CXCR3 in the kidneys of the conA-vehicle mice. Immunofluorescence microscopy demonstrated CXCR3 bound tightly to C-X-C motif ligand 11 (CXCL11) in the kidneys of the conA-vehicle mice and showed that PF treatment could suppress CXCR3/CXCL11 over-activation. Macrophage infiltration was a notable pathological change in the kidneys of conA-treated mice. PF administration attenuated conA-induced renal damage, at least in part, by inhibiting the over-activated CXCR3/CXCL11 signal axis.

  10. Radiation damage of gallium arsenide production cells

    NASA Technical Reports Server (NTRS)

    Mardesich, N.; Joslin, D.; Garlick, J.; Lillington, D.; Gillanders, M.; Cavicchi, B.; Scott-Monck, J.; Kachare, R.; Anspaugh, B.

    1987-01-01

    High efficiency liquid phase epitaxy (LPE) gallium arsenide cells were irradiated with 1 Mev electrons up to fluences of 1 times 10 to the 16th power cm-2. Measurements of spectral response and dark and illuminated I-V data were made at each fluence and then, using computer codes, the experimental data was fitted to gallium arsenide cell models. In this way it was possible to determine the extent of the damage, and hence damage coefficients in both the emitter and base of the cell.

  11. Cyanidin-3-O-beta-glucopyranoside, a natural free-radical scavenger against aflatoxin B1- and ochratoxin A-induced cell damage in a human hepatoma cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2).

    PubMed

    Guerra, M C; Galvano, F; Bonsi, L; Speroni, E; Costa, S; Renzulli, C; Cervellati, R

    2005-08-01

    Recent findings have suggested that oxidative damage might contribute to the cytotoxicity and carcinogenicity of aflatoxin B1 (AFB1). Induction of oxidative stress also plays an important role in the toxicity of another mycotoxin, ochratoxin A (OTA). In the present study, the protective effect of cyanidin-3-O-beta-glucopyranoside (C-3-G; an anthocyanin contained in oranges, blackberries, strawberries and cranberries) against AFB1- and OTA-induced cytotoxicity was investigated in a human hepatoma-derived cell line (Hep G2) and a human colonic adenocarcinoma cell line (CaCo-2). The ability of C-3-G to reduce the production of reactive oxygen species (ROS), the inhibition of protein and DNA synthesis and the apoptosis caused by the two mycotoxins was also investigated in both cell lines. Our experiments proved the significant cytoprotective effect of C-3-G in vitro against OTA- and AFB1-induced cell damage. In particular, 24 h of pretreatment with 50 microm-C-3-G inhibited the cytotoxicity of 10 microm-AFB1 (by 35 %) and of 10 microm-OTA (by 25 %) in Hep G2 cells (P < 0.001) and of 10 microm-AFB1 (by 10 %, P < 0.01) and of 10 microm-OTA (by 14 %, P < 0.05) in CaCo-2 cells. Moreover, 50 microm-C-3-G attenuated ROS production induced by the two toxins in both cell lines (P < 0.05). Inhibition of DNA and protein synthesis induced by the mycotoxins was counteracted by pretreatment with the antioxidant at 50 microm. Similarly, apoptotic cell death was prevented as demonstrated by a reduction of DNA fragmentation and inhibition of caspase-3 activation. The in vitro free-radical scavenging capacity of the anthocyanin was tested with the Briggs-Rauscher oscillating reaction. This system works at pH approximately 2. The results showed good scavenging power, in accordance with the observed inhibition of ROS production.

  12. Radiation Damage Workshop report. [solar cells

    NASA Technical Reports Server (NTRS)

    Rahilly, W. P.

    1980-01-01

    The starting material, cell design/geometry, and cell processing/fabrication for silicon and gallium arsenide solar cells are addressed with reference to radiation damage. In general, it is concluded that diagnostic sensitivities and material purities are basic to making significant gains in end-of-life performance and thermal annealability. Further, GaAs material characterization is so sketchy that a well defined program to evaluate such material for solar cell application is needed to maximize GaAs cell technology benefits.

  13. Myeloid-Derived Suppressor Cells Ameliorate Cyclosporine A-Induced Hypertension in Mice.

    PubMed

    Chiasson, Valorie L; Bounds, Kelsey R; Chatterjee, Piyali; Manandhar, Lochana; Pakanati, Abhinandan R; Hernandez, Marcos; Aziz, Bilal; Mitchell, Brett M

    2018-01-01

    The calcineurin inhibitor cyclosporine A (CsA) suppresses the immune system but promotes hypertension, vascular dysfunction, and renal damage. CsA decreases regulatory T cells and this contributes to the development of hypertension. However, CsA's effects on another important regulatory immune cell subset, myeloid-derived suppressor cells (MDSCs), is unknown. We hypothesized that augmenting MDSCs would ameliorate the CsA-induced hypertension and vascular and renal injury and dysfunction and that CsA reduces MDSCs in mice. Daily interleukin-33 treatment, which increased MDSC levels, completely prevented CsA-induced hypertension and vascular and renal toxicity. Adoptive transfer of MDSCs from control mice into CsA-treated mice after hypertension was established dose-dependently reduced blood pressure and vascular and glomerular injury. CsA treatment of aortas and kidneys isolated from control mice for 24 hours decreased relaxation responses and increased inflammation, respectively, and these effects were prevented by the presence of MDSCs. MDSCs also prevented the CsA-induced increase in fibronectin in microvascular and glomerular endothelial cells. Last, CsA dose-dependently reduced the number of MDSCs by inhibiting calcineurin and preventing cell proliferation, as other direct calcineurin signaling pathway inhibitors had the same dose-dependent effect. These data suggest that augmenting MDSCs can reduce the cardiovascular and renal toxicity and hypertension caused by CsA. © 2017 American Heart Association, Inc.

  14. Alcohol Injury Damages Intestinal Stem Cells.

    PubMed

    Lu, Rong; Voigt, Robin M; Zhang, Yongguo; Kato, Ikuko; Xia, Yinglin; Forsyth, Christopher B; Keshavarzian, Ali; Sun, Jun

    2017-04-01

    Alcohol consumption is associated with intestinal injury including intestinal leakiness and the risk of developing progressive gastrointestinal cancer. Alcoholics have disruption of intestinal barrier dysfunction that persists weeks after stopping alcohol intake, and this occurs in spite of the fact that intestinal epithelial cells turn over every 3 to 5 days. The renewal and functional regulation of the intestinal epithelium largely relies on intestinal stem cells (ISCs). Chronic inflammation and tissue damage in the intestine can injure stem cells including accumulation of mutations that may result in ISC dysfunction and transformation. ISCs are a key element in intestinal function and pathology; however, very little is known about the effects of alcohol on ISCs. We hypothesize that dysregulation of ISCs is one mechanism by which alcohol induces long-lasting intestinal damage. In Vivo: Small intestinal samples from alcohol- and control-fed mice were assessed for ISC markers (Lgr5 and Bmi1) and the changes of the β-catenin signaling using immunofluorescent microscopy, Western blotting, and RT-PCR. Ex Vivo: Organoids were generated from small intestine tissue and subsequently exposed to alcohol and analyzed for ISC markers, β-catenin signaling. Chronic alcohol consumption significantly decreased the expression of stem cell markers, Bmi1 in the small intestine of the alcohol-fed mice and also resulted in dysregulation of the β-catenin signaling-an essential regulator of its target gene Lgr5 and ISC function. Exposure of small intestine-derived organoids to 0.2% alcohol significantly reduced the growth of the organoids, including budding, and total surface area of the organoid cultures. Alcohol also significantly decreased the expression of Lgr5, p-β-catenin (ser552), and Bmi1 in the organoid model. Both chronic alcohol feeding and acute exposure of alcohol resulted in ISC dysregulation which might be one mechanism for alcohol-induced long-lasting intestinal

  15. Helicobacter pylori VacA induces autophagic cell death in gastric epithelial cells via the endoplasmic reticulum stress pathway.

    PubMed

    Zhu, Pan; Xue, Jun; Zhang, Zhu-Jun; Jia, Yin-Ping; Tong, Ya-Nan; Han, Dan; Li, Qian; Xiang, Yang; Mao, Xu-Hu; Tang, Bin

    2017-12-13

    The Helicobacter pylori vacuolating cytotoxin (VacA) can promote progressive vacuolation and gastric injury and may be associated with human gastric cancer. Increasing evidence indicates that autophagy is involved in the cell death induced by VacA, but the specific mechanisms need to be further elucidated. We show here that VacA could induce autophagy and increase cell death in human gastric cancer cell lines. Further investigations revealed that inhibition of autophagy could decrease the VacA-induced cell death in AGS cells. Furthermore, numerous dilated endoplasmic reticula (ER) were observed, and the phosphorylation of a subunit of eukaryotic translation initiation factor 2 subunit 1 also increased in the VacA-treated AGS cells, while repression of ER stress could reduce autophagy and cell death through knockdown of activating transcription factor 4 and DNA-damage-inducible transcript 3. In addition, the expression of pseudokinase tribbles homolog 3 (TRIB3) upon ER stress was triggered by VacA, and knockdown of TRIB3 could also decrease VacA-induced cell death. Finally, inhibition of autophagy could decrease VacA s1m1 -induced cell death and apoptosis, and apoptosis inhibitor Z-VAD had no significant effect on autophagy induced by VacA s1m1 . Thus, these results suggested that VacA causes autophagic cell death via ER stress in gastric epithelial cells.

  16. Space solar cells: High efficiency and radiation damage

    NASA Technical Reports Server (NTRS)

    Brandhorst, H., Jr.; Bernatowicz, D. T.

    1980-01-01

    The progress and status of efforts to increase the end-of-life efficiency of solar cells for space use is assessed. High efficiency silicon solar cells, silicon solar cell radiation damage, GaAs solar cell performance and radiation damage and 30 percent devices are discussed.

  17. DNA damage checkpoints in stem cells, ageing and cancer.

    PubMed

    Sperka, Tobias; Wang, Jianwei; Rudolph, K Lenhard

    2012-09-01

    DNA damage induces cell-intrinsic checkpoints, including p53 and retinoblastoma (RB), as well as upstream regulators (exonuclease 1 (EXO1), ataxia telangiectasia mutated (ATM), ATR, p16(INK4a) and p19(ARF)) and downstream targets (p21, PUMA (p53 upregulated modulator of apoptosis) and sestrins). Clearance of damaged cells by cell-intrinsic checkpoints suppresses carcinogenesis but as a downside may impair stem cell and tissue maintenance during ageing. Modulating the activity of DNA damage checkpoints can either accelerate or decelerate tissue ageing and age-related carcinogenesis. The outcome depends on cell-intrinsic and cell-extrinsic mechanisms that regulate the clearance of damaged cells and on the molecular context in ageing tissues, including the level of DNA damage accumulation itself.

  18. Laser damage resistant nematic liquid crystal cell

    NASA Astrophysics Data System (ADS)

    Raszewski, Z.; Piecek, W.; Jaroszewicz, L.; Soms, L.; Marczak, J.; Nowinowski-Kruszelnicki, E.; Perkowski, P.; Kedzierski, J.; Miszczyk, E.; Olifierczuk, M.; Morawiak, P.; Mazur, R.

    2013-08-01

    There exists a problem in diagnostics of a dense plasma (so-called Thomson diagnostics). For this purpose, the plasma is illuminated by series of high energy laser pulses. Such pulses are generated by several independent lasers operating sequentially, and these pulses are to be directed along an exactly the same optical path. In this case, the energy of each separate pulse is as large as 3 J, so it is impossible to generate a burst of such pulses by a single laser. In this situation, several independent lasers have to be used. To form optical path with λ = 1.064 μm and absolute value of the energy of laser pulse through of 3 J, a special refractive index matched twisted Nematic Liquid Crystal Cell (NLCC) of type LCNP2 with switching on time τON smaller than 5 μs might be applied. High laser damage resistance of NLCC and short τON can be fulfilled by preparation of liquid crystal cells with Liquid Crystal Mixture (LCM), well tuned to twisted nematic electro-optical effect, and well tuned all optical interfaces (Air - Antireflection - Quartz Plate - Electrode - Blocking Film - Aligning Layer - LCM - Aligning Layer - Blocking Film - Electrode - Quartz Plate - Antireflection - Air). In such LCNP2 cell, the transmission is higher than 97% at λ = 1.064 μm, as it is presented by Gooch and Tarry [J. Phys. D: Appl. Phys. 8, 1575 (1975)]. The safe laser density energy is about 0.6 J/cm2 for a train of laser pulses (λ = 1.064 μm, pulse duration 10 ns FWHM, pulse repetition rate 100 pps, train duration 10 s), so the area of liquid crystal cell tolerating 3 J through it shall be as large as 5 cm2. Due to the presence of two blocking film layers between electrodes, LCNP2 can be driven by high voltages. Switching on time smaller than τON = 5 μs was obtained under 200 V switching voltage.

  19. Protective effect of Cordyceps militaris extract against bisphenol A induced reproductive damage.

    PubMed

    Wang, Jian; Chen, Chen; Jiang, Zhihui; Wang, Meng; Jiang, Hai; Zhang, Xiaoying

    2016-08-01

    This study aimed to investigate the protective effects of Cordyceps militaris (C. militaris) against reproductive damage induced by bisphenol A (BPA). Rats were administrated 200 mg/kg BPA for 4 weeks and treated with C. militaris (200, 400, and 800 mg/kg body weight/day). By the end of the fourth week, the level of oxidative damage, sperm parameters, hormone levels, and histopathological changes were examined. In the group that only received BPA, there was a significant decrease in body weight compared with the normal control (NC) group. C. militaris significantly alleviated the BPA-induced reproductive damage by increasing testicular superoxide dismutase (SOD), glutathione peroxidase (GSH-PX), and glutathione (GSH); as well as by reducing serum malondialdehyde (MDA). C. militaris not only obviously enhanced the levels of serum LH and T, but it also improved the sperm count and motility compared to the BPA-treated group. These results suggest that C. militaris could be used as a potential natural substance for preventing BPA induced reproductive damage. Abbreviations BPA: bisphenol A; SOD: superoxide dismutase; GSH: glutathione; GSH-PX: glutathione peroxidase; MDA: malondialdehyde; ROS: reactive oxygen species; T: testosterone; LH: luteinizing hormone; FSH: follicle-stimulating hormone; UPLC: ultra performance liquid chromatography; RIA: radioimmunoassay; q quantitative real time PCR; NC: normal control group; BPA: 200 mg/kg BPA administered group; H: 800 mg/kg C. militaris extract administered group; LB, MB, and HB: 200 mg/kg BPA + 200 mg/kg, 400 mg/kg, and 800 mg/kg C. militaris administered group, respectively; VeB: 200 mg/kg BPA + 300 mg/kg Vitamin E administered group; Star: steroidogenic acute regulatory protein; 3β-HSD: 3beta-hydroxyl-delta-5-steroid dehydrogenase; CYP11A1: cytochrome P 450 family 11 subfamily A member 1; CYP17A1: cytochrome P 450 family 17 subfamily A member 1.

  20. Predictions of cell damage rates for Lifesat missions

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Atwell, William; Hardy, Alva C.; Golightly, Michael J.; Wilson, John W.; Townsend, Lawrence W.; Shinn, Judy; Nealy, John E.; Katz, Robert

    1990-01-01

    The track model of Katz is used to make predictions of cell damage rates for possible Lifesat experiments. Contributions from trapped protons and electrons and galactic cosmic rays are considered for several orbits. Damage rates for survival and transformation of C3HT10-1/2 cells are predicted for various spacecraft shields.

  1. Cell damage evaluation of mammalian cells in cell manipulation by amplified femtosecond ytterbium laser

    NASA Astrophysics Data System (ADS)

    Hong, Z.-Y.; Iino, T.; Hagihara, H.; Maeno, T.; Okano, K.; Yasukuni, R.; Hosokawa, Y.

    2018-03-01

    A micrometer-scale explosion with cavitation bubble generation is induced by focusing a femtosecond laser in an aqueous solution. We have proposed to apply the explosion as an impulsive force to manipulate mammalian cells especially in microfluidic chip. Herein, we employed an amplified femtosecond ytterbium laser as an excitation source for the explosion and evaluated cell damage in the manipulation process to clarify the application potential. The damage of C2C12 myoblast cell prepared as a representative mammalian cell was investigated as a function of distance between cell and laser focal point. Although the cell received strong damage on the direct laser irradiation condition, the damage sharply decreased with increasing distance. Since the threshold distance, above which the cell had no damage, was consistent with radius of the cavitation bubble, impact of the cavitation bubble would be a critical factor for the cell damage. The damage had strong nonlinearity in the pulse energy dependence. On the other hand, cell position shift by the impact of the cavitation bubble was almost proportional to the pulse energy. In balance between the cell viability and the cell position shift, we elucidated controllability of the cell manipulation in microfluidic chip.

  2. Tool permits damage-free removal of solar cell

    NASA Technical Reports Server (NTRS)

    Beckley, J. E., Jr.

    1966-01-01

    Modified soldering iron extracts a wrap-around solar cell that is attached with solder or adhesive to a substrate without destroying the cell removed or damaging adjacent cells. Heat, vacuum, and compressed air, operated from a special head attached to the soldering iron, loosen, extract, and protect the cell.

  3. Track structure model of cell damage in space flight

    NASA Technical Reports Server (NTRS)

    Katz, Robert; Cucinotta, Francis A.; Wilson, John W.; Shinn, Judy L.; Ngo, Duc M.

    1992-01-01

    The phenomenological track-structure model of cell damage is discussed. A description of the application of the track-structure model with the NASA Langley transport code for laboratory and space radiation is given. Comparisons to experimental results for cell survival during exposure to monoenergetic, heavy-ion beams are made. The model is also applied to predict cell damage rates and relative biological effectiveness for deep-space exposures.

  4. DNA damage in cells exhibiting radiation-induced genomic instability

    DOE PAGES

    Keszenman, Deborah J.; Kolodiuk, Lucia; Baulch, Janet E.

    2015-02-22

    Cells exhibiting radiation induced genomic instability exhibit varied spectra of genetic and chromosomal aberrations. Even so, oxidative stress remains a common theme in the initiation and/or perpetuation of this phenomenon. Isolated oxidatively modified bases, abasic sites, DNA single strand breaks and clustered DNA damage are induced in normal mammalian cultured cells and tissues due to endogenous reactive oxygen species generated during normal cellular metabolism in an aerobic environment. While sparse DNA damage may be easily repaired, clustered DNA damage may lead to persistent cytotoxic or mutagenic events that can lead to genomic instability. In this study, we tested the hypothesismore » that DNA damage signatures characterised by altered levels of endogenous, potentially mutagenic, types of DNA damage and chromosomal breakage are related to radiation-induced genomic instability and persistent oxidative stress phenotypes observed in the chromosomally unstable progeny of irradiated cells. The measurement of oxypurine, oxypyrimidine and abasic site endogenous DNA damage showed differences in non-double-strand breaks (DSB) clusters among the three of the four unstable clones evaluated as compared to genomically stable clones and the parental cell line. These three unstable clones also had increased levels of DSB clusters. The results of this study demonstrate that each unstable cell line has a unique spectrum of persistent damage and lead us to speculate that alterations in DNA damage signaling and repair may be related to the perpetuation of genomic instability.« less

  5. Quantifying Low Energy Proton Damage in Multijunction Solar Cells

    NASA Technical Reports Server (NTRS)

    Messenger, Scott R.; Burke, Edward A.; Walters, Robert J.; Warner, Jeffrey H.; Summers, Geoffrey P.; Lorentzen, Justin R.; Morton, Thomas L.; Taylor, Steven J.

    2007-01-01

    An analysis of the effects of low energy proton irradiation on the electrical performance of triple junction (3J) InGaP2/GaAs/Ge solar cells is presented. The Monte Carlo ion transport code (SRIM) is used to simulate the damage profile induced in a 3J solar cell under the conditions of typical ground testing and that of the space environment. The results are used to present a quantitative analysis of the defect, and hence damage, distribution induced in the cell active region by the different radiation conditions. The modelling results show that, in the space environment, the solar cell will experience a uniform damage distribution through the active region of the cell. Through an application of the displacement damage dose analysis methodology, the implications of this result on mission performance predictions are investigated.

  6. Mechanisms of cell damage in agitated microcarrier tissue culture reactors

    NASA Technical Reports Server (NTRS)

    Cherry, Robert S.; Papoutsakis, E. Terry

    1986-01-01

    Cells growing on microcarriers may be damaged by collisions of the microcarrier against another microcarrier or the reactor agitator. Bead-bead collisions are caused by small-scale turbulence, which can also cause high local shear stress on the cells. The cells are also exposed to 10-20 Hz cyclic shear stress by bead rotation.

  7. Novel DNA damage checkpoint in mitosis: Mitotic DNA damage induces re-replication without cell division in various cancer cells.

    PubMed

    Hyun, Sun-Yi; Rosen, Eliot M; Jang, Young-Joo

    2012-07-06

    DNA damage induces multiple checkpoint pathways to arrest cell cycle progression until damage is repaired. In our previous reports, when DNA damage occurred in prometaphase, cells were accumulated in 4 N-DNA G1 phase, and mitosis-specific kinases were inactivated in dependent on ATM/Chk1 after a short incubation for repair. We investigated whether or not mitotic DNA damage causes cells to skip-over late mitotic periods under prolonged incubation in a time-lapse study. 4 N-DNA-damaged cells re-replicated without cell division and accumulated in 8 N-DNA content, and the activities of apoptotic factors were increased. The inhibition of DNA replication reduced the 8 N-DNA cell population dramatically. Induction of replication without cell division was not observed upon depletion of Chk1 or ATM. Finally, mitotic DNA damage induces mitotic slippage and that cells enter G1 phase with 4 N-DNA content and then DNA replication is occurred to 8 N-DNA content before completion of mitosis in the ATM/Chk1-dependent manner, followed by caspase-dependent apoptosis during long-term repair. Copyright © 2012 Elsevier Inc. All rights reserved.

  8. Gallium Arsenide solar cell radiation damage experiment

    NASA Technical Reports Server (NTRS)

    Maurer, R. H.; Kinnison, J. D.; Herbert, G. A.; Meulenberg, A.

    1991-01-01

    Gallium arsenide (GaAs) solar cells for space applications from three different manufactures were irradiated with 10 MeV protons or 1 MeV electrons. The electrical performance of the cells was measured at several fluence levels and compared. Silicon cells were included for reference and comparison. All the GaAs cell types performed similarly throughout the testing and showed a 36 to 56 percent power areal density advantage over the silicon cells. Thinner (8-mil versus 12-mil) GaAs cells provide a significant weight reduction. The use of germanium (Ge) substrates to improve mechanical integrity can be implemented with little impact on end of life performance in a radiation environment.

  9. Quantitative Profiling of DNA Damage and Apoptotic Pathways in UV Damaged Cells Using PTMScan Direct

    PubMed Central

    Stokes, Matthew P.; Silva, Jeffrey C.; Jia, Xiaoying; Lee, Kimberly A.; Polakiewicz, Roberto D.; Comb, Michael J.

    2013-01-01

    Traditional methods for analysis of peptides using liquid chromatography and tandem mass spectrometry (LC-MS/MS) lack the specificity to comprehensively monitor specific biological processes due to the inherent duty cycle limitations of the MS instrument and the stochastic nature of the analytical platform. PTMScan Direct is a novel, antibody-based method that allows quantitative LC-MS/MS profiling of specific peptides from proteins that reside in the same signaling pathway. New PTMScan Direct reagents have been produced that target peptides from proteins involved in DNA Damage/Cell Cycle and Apoptosis/Autophagy pathways. Together, the reagents provide access to 438 sites on 237 proteins in these signaling cascades. These reagents have been used to profile the response to UV damage of DNA in human cell lines. UV damage was shown to activate canonical DNA damage response pathways through ATM/ATR-dependent signaling, stress response pathways and induce the initiation of apoptosis, as assessed by an increase in the abundance of peptides corresponding to cleaved, activated caspases. These data demonstrate the utility of PTMScan Direct as a multiplexed assay for profiling specific cellular responses to various stimuli, such as UV damage of DNA. PMID:23344034

  10. Fatigue and Mechanical Damage Propagation in Automotive PEM Fuel Cells

    NASA Astrophysics Data System (ADS)

    Banan, Roshanak

    Polymer electrolyte membrane (PEM) fuel cells are generally exposed to high magnitude road-induced vibrations and impact loads, frequent humidity-temperature loading cycles, and freeze/thaw stresses when employed in automotive applications. The resultant mechanical stresses can play a significant role in the evolution of mechanical defects in the membrane electrode assembly (MEA). The focus of this research is to investigate fatigue challenges due to humidity-temperature (hygrothermal) cycles and vibrations and their effects on damage evolution in PEM fuel cells. To achieve this goal, this thesis is divided into three parts that provide insight into damage propagation in the MEA under i) hygrothermal cycles, ii) external applied vibrations, and iii) a combination of both to simulate realistic automotive conditions. A finite element damage model based on cohesive zone theory was developed to simulate the propagation of micro-scale defects (cracks and delaminations) in the MEA under fuel cell operating conditions. It was found that the micro-defects can propagate to critical states under start-up and shut-down cycles, prior to reaching the desired lifespan of the fuel cell. The simultaneous presence of hygrothermal cycles and vibrations severely intensified damage propagation and resulted in considerably large defects within 75% of the fuel cell life expectancy. However, the order of generated damage was found to be larger under hygrothermal cycles than vibrations. Under hygrothermal cycles, membrane crack propagation was more severe compared to delamination propagation. Conversely, the degrading influence of vibrations was more significant on delaminations. The presence of an anode/cathode channel offset under the combined loadings lead to a 2.5-fold increase in the delamination length compared to the aligned-channel case. The developed model can be used to investigate the damage behaviour of current materials employed in fuel cells as well as to evaluate the

  11. Electron Radiation Damage of (alga) As-gaas Solar Cells

    NASA Technical Reports Server (NTRS)

    Loo, R.; Kamath, G. S.; Knechtli, R.

    1979-01-01

    Solar cells (2 cm by 2 cm (AlGa) As-GaAs cells) were fabricated and then subjected to irradiation at normal incidence by electrons. The influence of junction depth and n-type buffer layer doping level on the cell's resistance to radiation damage was investigated. The study shows that (1) a 0.3 micrometer deep junction results in lower damage to the cells than does a 0.5 micrometer junction, and (2) lowering the n buffer layer doping density does not improve the radiation resistance of the cell. Rather, lowering the doping density decreases the solar cell's open circuit voltage. Some preliminary thermal annealing experiments in vacuum were performed on the (AlGa)As-GaAs solar cells damaged by 1-MeV electron irradiation. The results show that cell performance can be expected to partially recover at 200 C with more rapid and complete recovery occurring at higher temperature. For a 0.5hr anneal at 400 C, 90% of the initial power is recovered. The characteristics of the (AlGa)As-GaAs cells both before and after irradiation are described.

  12. Hydroxytyrosol Protects against Oxidative DNA Damage in Human Breast Cells

    PubMed Central

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J.

    2011-01-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol’s effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells. PMID:22254082

  13. Hydroxytyrosol protects against oxidative DNA damage in human breast cells.

    PubMed

    Warleta, Fernando; Quesada, Cristina Sánchez; Campos, María; Allouche, Yosra; Beltrán, Gabriel; Gaforio, José J

    2011-10-01

    Over recent years, several studies have related olive oil ingestion to a low incidence of several diseases, including breast cancer. Hydroxytyrosol and tyrosol are two of the major phenols present in virgin olive oils. Despite the fact that they have been linked to cancer prevention, there is no evidence that clarifies their effect in human breast tumor and non-tumor cells. In the present work, we present hydroxytyrosol and tyrosol's effects in human breast cell lines. Our results show that hydroxytyrosol acts as a more efficient free radical scavenger than tyrosol, but both fail to affect cell proliferation rates, cell cycle profile or cell apoptosis in human mammary epithelial cells (MCF10A) or breast cancer cells (MDA-MB-231 and MCF7). We found that hydroxytyrosol decreases the intracellular reactive oxygen species (ROS) level in MCF10A cells but not in MCF7 or MDA-MB-231 cells while very high amounts of tyrosol is needed to decrease the ROS level in MCF10A cells. Interestingly, hydroxytyrosol prevents oxidative DNA damage in the three breast cell lines. Therefore, our data suggest that simple phenol hydroxytyrosol could contribute to a lower incidence of breast cancer in populations that consume virgin olive oil due to its antioxidant activity and its protection against oxidative DNA damage in mammary cells.

  14. Multistage carcinogenesis modeling including cell cycle and DNA damage states

    NASA Astrophysics Data System (ADS)

    Hazelton, W.; Moolgavkar, S.

    The multistage clonal expansion model of carcinogenesis is generalized to include cell cycle states and corresponding DNA damage states with imperfect repair for normal and initiated stem cells. Initiated cells may undergo transformation to a malignant state, eventually leading to cancer incidence or death. The model allows oxidative or radiation induced DNA damage, checkpoint delay, DNA repair, apoptosis, and transformation rates to depend on the cell cycle state or DNA damage state of normal and initiated cells. A probability generating function approach is used to represent the time dependent probability distribution for cells in all states. The continuous time coupled Markov system representing this joint distribution satisfies a partial differential equation (pde). Time dependent survival and hazard functions are found through numerical solution of the characteristic equations for the pde. Although the hazard and survival can be calculated numerically, number and size distributions of pre-malignant lesions from models that are developed will be approximated through simulation. We use the model to explore predictions for hazard and survival as parameters representing cell cycle regulation and arrest are modified. Modification of these parameters may influence rates for cell division, apoptosis and malignant transformation that are important in carcinogenesis. We also explore enhanced repair that may be important for low-dose hypersensitivity and adaptive response, and degradation of repair processes or loss of checkpoint control that may drive genetic instability.

  15. Anaphylatoxin C3a induced mediator release from mast cells

    SciTech Connect

    Herrscher, R.; Hugli, T.E.; Sullivan, T.J.

    1986-03-01

    The authors investigated the biochemical and functional consequences of the binding of highly purified human C3a to isolated rat serosal mast cells. C3a caused a dose-dependent (1-30 ..mu..M), noncytotoxic release of up to 64% (+/- 7 SEM) of the mast cell histamine content. C3a (10..mu..M) increased /sup 45/Ca/sup + +/ uptake 8.2- fold (+/- 2.2 SEM) above unstimulated control values within 10 minutes. Arachidonyl-diacylglycerol and arachidonyl-monoacylglycerol levels increased significantly within 2 minutes after C3a (10 ..mu..M) stimulation. Turnover of phosphatidylinositol, phosphatidic acid, and phosphatidylcholine were increased within 15 minutes. In contrast to antigen, C3a stimulation (10 ..mu..M) was not enhancedmore » by exogenous phosphatidylserine, and was not inhibited by ethanol (100 ..mu..mM). C3a suppressed arachidonic acid (AA) release to 38% (+/- 9 SEM) below baseline, and did not cause PGD/sub 2/ formation. C3a and the desarginine form of C3a caused identical responses in all experiments. These studies indicate that C3a stimulation activates mast cell preformed mediator release in a manner very similar to antigen-IgE stimulation, but C3a suppresses free AA levels and does not stimulate PGD/sub 2/ synthesis.« less

  16. γδ T cells are indispensable for interleukin-23-mediated protection against Concanavalin A-induced hepatitis in hepatitis B virus transgenic mice.

    PubMed

    Meng, Ziyu; Wang, Jingya; Yuan, Yifang; Cao, Guangchao; Fan, Shuobing; Gao, Chao; Wang, Li; Li, Zheng; Wu, Xiaoli; Wu, Zhenzhou; Zhao, Liqing; Yin, Zhinan

    2017-05-01

    Hepatitis B virus surface antigen (HBsAg) carriers are highly susceptible to liver injury triggered by environmental biochemical stimulation. Previously, we have reported an inverse correlation between γδ T cells and liver damage in patients with hepatitis B virus (HBV). However, whether γδ T cells play a role in regulating the hypersensitivity of HBsAg carriers to biochemical stimulation-induced hepatitis is unknown. In this study, using HBV transgenic (HBs-Tg) and HBs-Tg T-cell receptor-δ-deficient (TCR-δ -/- ) mice, we found that mice genetically deficient in γδ T cells exhibited more severe liver damage upon Concanavalin A (Con A) treatment, as indicated by substantially higher serum alanine aminotransferase levels, further elevated interferon-γ (IFN-γ) levels and more extensive necrosis. γδ T-cell deficiency resulted in elevated IFN-γ in CD4 + T cells but not in natural killer or natural killer T cells. The depletion of CD4 + T cells and neutralization of IFN-γ reduced liver damage in HBs-Tg and HBs-Tg-TCR-δ -/- mice to a similar extent. Further investigation revealed that HBs-Tg mice showed an enhanced interleukin-17 (IL-17) signature. The administration of exogenous IL-23 enhanced IL-17A production from Vγ4 γδ T cells and ameliorated liver damage in HBs-Tg mice, but not in HBs-Tg-TCR-δ -/- mice. In summary, our results demonstrated that γδ T cells played a protective role in restraining Con A-induced hepatitis by inhibiting IFN-γ production from CD4 + T cells and are indispensable for IL-23-mediated protection against Con A-induced hepatitis in HBs-Tg mice. These results provided a potential therapeutic approach for treating the hypersensitivity of HBV carriers to biochemical stimulation-induced liver damage. © 2017 John Wiley & Sons Ltd.

  17. Quantifying the potential for bursting bubbles to damage suspended cells.

    PubMed

    Walls, Peter L L; McRae, Oliver; Natarajan, Venkatesh; Johnson, Chris; Antoniou, Chris; Bird, James C

    2017-11-08

    Bubbles that rise to the surface of a cell suspension can damage cells when they pop. This phenomenon is particularly problematic in the biotechnology industry, as production scale bioreactors require continuous injection of oxygen bubbles to maintain cell growth. Previous studies have linked cell damage to high energy dissipation rates (EDR) and have predicted that for small bubbles the EDR could exceed values that would kill many cells used in bioreactors, including Chinese Hamster Ovary (CHO) cells. However, it's unclear how many cells would be damaged by a particular bursting bubble, or more precisely how much volume around the bubble experiences these large energy dissipation rates. Here we quantify these volumes using numerical simulations and demonstrate that even though the volume exceeding a particular EDR increases with bubble size, on a volume-to-volume basis smaller bubbles have a more significant impact. We validate our model with high-speed experiments and present our results in a non-dimensionalized framework, enabling predictions for a variety of liquids and bubble sizes. The results are not restricted to bubbles in bioreactors and may be relevant to a variety of applications ranging from fermentation processes to characterizing the stress levels experienced by microorganisms within the sea surface microlayer.

  18. Bacterial Cell Surface Damage Due to Centrifugal Compaction

    PubMed Central

    Peterson, Brandon W.; van der Mei, Henny C.; Busscher, Henk J.

    2012-01-01

    Centrifugal damage has been known to alter bacterial cell surface properties and interior structures, including DNA. Very few studies exist on bacterial damage caused by centrifugation because of the difficulty in relating centrifugation speed and container geometry to the damage caused. Here, we provide a simple, versatile method of analysis for describing the compaction of bacteria during centrifugation based on a proposed centrifugation coefficient, C. Values of C can be related to different bacterial cell surface properties. Changing the geometry of the centrifugation container or centrifugation speeds changed the value of C significantly. Initial deposition rates of Staphylococcus aureus ATCC 12600 to a glass surface decayed exponentially from 4,217 to 1,478 cm−2 s−1 with increasing C, while the proportion of staphylococci with a zeta potential of around −15 mV decreased from 97 to 58%. These surface-sensitive parameters were used independently to derive a critical centrifugation coefficient (0.040), above which centrifugation was considered to impact the outcome of surface-sensitive experiments due to cell surface damage. The critical centrifugation coefficient could successfully predict staphylococcal cell surface damage, i.e., a significant change in initial deposition rate or zeta potential distribution, in 84% of all cases included here, whereas the centrifugation speed could predict damage in only 58% of all cases. Moreover, controlling the centrifugation coefficient within narrow limits over a series of experiments yielded 43% smaller standard deviations in initial staphylococcal deposition rates than with centrifugation at fixed speeds for replicate experiments. PMID:22038609

  19. Cell-Based Memory of DNA Damage in Breast Cancer

    DTIC Science & Technology

    2009-09-01

    them into tumor cells. 15. SUBJECT TERMS synthetic biology , cancer progression, initiation, DNA damage, genetic circuits 16. SECURITY...unique tuning device using miRNAs. Taken together, these results lay at the foundation of the application of Synthetic Biology to cancer studies and

  20. 34. Historic photo of Building 202 test cell with damage ...

    Library of Congress Historic Buildings Survey, Historic Engineering Record, Historic Landscapes Survey

    34. Historic photo of Building 202 test cell with damage from fire or explosion during rocket engine testing, May 17, 1958. On file at NASA Plumbrook Research Center, Sandusky, Ohio. NASA photo number C-47965. - Rocket Engine Testing Facility, GRC Building No. 202, NASA Glenn Research Center, Cleveland, Cuyahoga County, OH

  1. Targeting Werner syndrome protein sensitizes U-2 OS osteosarcoma cells to selenium-induced DNA damage response and necrotic death.

    PubMed

    Cheng, Wen-Hsing; Wu, Ryan T Y; Wu, Min; Rocourt, Caroline R B; Carrillo, Jose A; Song, Jiuzhou; Bohr, Christina T; Tzeng, Tiffany J

    2012-03-30

    Mutations in the Werner syndrome protein (WRN), a caretaker of the genome, result in Werner syndrome, which is characterized by premature aging phenotypes and cancer predisposition. Methylseleninic acid (MSeA) can activate DNA damage responses and is a superior compound to suppress tumorigenesis in mouse models of cancer. To test the hypothesis that targeting WRN can potentiate selenium toxicity in cancer cells, isogenic WRN small hairpin RNA (shRNA) and control shRNA U-2 OS osteosarcoma cells were treated with MSeA for 2d, followed by recovery for up to 7d. WRN deficiency sensitized U-2 OS cells to MSeA-induced necrotic death. Co-treatment with the ataxia-telangiectasia mutated (ATM) kinase inhibitor KU55933 desensitized the control shRNA cells, but not WRN shRNA cells, to MSeA treatment. WRN did not affect MSeA-induced ATM phosphorylation on Ser-1981 or H2A.X phosphorylation on Ser-139, but promoted recovery from the MSeA-induced DNA damage. Taken together, WRN protects U-2 OS osteosarcoma cells against MSeA-induced cytotoxicity, suggesting that oxidative DNA repair pathway is a promising target for improving the efficacy of selenium on tumor suppression. Copyright © 2012 Elsevier Inc. All rights reserved.

  2. A new cell cycle checkpoint that senses plasma membrane/cell wall damage in budding yeast.

    PubMed

    Kono, Keiko; Ikui, Amy E

    2017-04-01

    In nature, cells face a variety of stresses that cause physical damage to the plasma membrane and cell wall. It is well established that evolutionarily conserved cell cycle checkpoints monitor various cellular perturbations, including DNA damage and spindle misalignment. However, the ability of these cell cycle checkpoints to sense a damaged plasma membrane/cell wall is poorly understood. To the best of our knowledge, our recent paper described the first example of such a checkpoint, using budding yeast as a model. In this review, we will discuss this important question as well as provide hypothetical explanations to be tested in the future. © 2017 WILEY Periodicals, Inc.

  3. Arctigenin protects against ultraviolet-A-induced damage to stemness through inhibition of the NF-κB/MAPK pathway.

    PubMed

    Park, See-Hyoung; Cho, Jae Youl; Oh, Sae Woong; Kang, Mingyeong; Lee, Seung Eun; Yoo, Ju Ah; Jung, Kwangseon; Lee, Jienny; Lee, Sang Yeol; Lee, Jongsung

    2018-02-25

    The stemness of stem cells is negatively affected by ultraviolet A (UVA) irradiation. This study was performed to examine the effects of arctigenin on UVA-irradiation-induced damage to the stemness of human mesenchymal stem cells (hMSCs) derived from adipose tissue. The mechanisms of action of arctigenin were also investigated. A BrdU-incorporation assay demonstrated that arctigenin attenuated the UVA-induced reduction of the cellular proliferative potential. Arctigenin also increased the UVA-induced reduction in stemness of hMSCs by upregulating stemness-related genes such as SOX2, OCT4, and NANOG. In addition, the UVA-induced reduction in the mRNA expression level of hypoxia-inducible factor (HIF)-1α was significantly recovered by arctigenin. The antagonizing effect of arctigenin on UVA irradiation was mediated by reduced PGE 2 production through the inhibition of MAPKs (p42/44 MAPK, p38 MAPK, and JNK) and NF-κB. Overall, these findings suggest that arctigenin can ameliorate the reduced stemness of hMSCs induced by UVA irradiation. The effects of arctigenin are mediated by PGE 2 -cAMP signaling-dependent upregulation of HIF-1α. Therefore, arctigenin could be used as an antagonist to attenuate the effects of UVA irradiation. Copyright © 2018 Elsevier B.V. All rights reserved.

  4. Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage.

    PubMed

    Yu, Zhiliang; Cai, Yuanning; Qin, Wangrong; Lin, Jianxun; Qiu, Juanping

    2015-01-01

    Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg2+, Ca2+ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa.

  5. Polymyxin E Induces Rapid Paenibacillus polymyxa Death by Damaging Cell Membrane while Ca2+ Can Protect Cells from Damage

    PubMed Central

    Cai, Yuanning; Qin, Wangrong; Lin, Jianxun

    2015-01-01

    Polymyxin E, produced by Paenibacillus polymyxa, is an important antibiotic normally against Gram-negative pathogens. In this study, we found that polymyxin E can kill its producer P. polymyxa, a Gram-positive bacterium, by disrupting its cell membrane. Membrane damage was clearly revealed by detecting the leakage of intracellular molecules. The observation using scanning electron microscopy also supported that polymyxin E can destroy the cell membrane and cause an extensive cell surface alteration. On the other hand, divalent cations can give protection against polymyxin E. Compared with Mg2+, Ca2+ can more effectively alleviate polymyxin E-induced damage to the cell membrane, thus remarkably increasing the P. polymyxa survival. Our findings would shed light on a not yet described bactericidal mechanism of polymyxin E against Gram-positive bacteria and more importantly the nature of limited fermentation output of polymyxin E from P. polymyxa. PMID:26252512

  6. Multiscale modelling of Flow-Induced Blood Cell Damage

    NASA Astrophysics Data System (ADS)

    Liu, Yaling; Sohrabi, Salman

    2017-11-01

    We study red blood cell (RBC) damage and hemolysis at cellular level. Under high shear rates, pores form on RBC membranes through which hemoglobin (Hb) leaks out and increases free Hb content of plasma leading to hemolysis. By coupling lattice Boltzmann and spring connected network models through immersed boundary method, we estimate hemolysis of a single RBC under various shear rates. The developed cellular damage model can be used as a predictive tool for hydrodynamic and hematologic design optimization of blood-wetting medical devices.

  7. Thermal effects in Cs DPAL and alkali cell window damage

    NASA Astrophysics Data System (ADS)

    Zhdanov, B. V.; Rotondaro, M. D.; Shaffer, M. K.; Knize, R. J.

    2016-10-01

    Experiments on power scaling of Diode Pumped Alkali Lasers (DPALs) revealed some limiting parasitic effects such as alkali cell windows and gain medium contamination and damage, output power degradation in time and others causing lasing efficiency decrease or even stop lasing1 . These problems can be connected with thermal effects, ionization, chemical interactions between the gain medium components and alkali cells materials. Study of all these and, possibly, other limiting effects and ways to mitigate them is very important for high power DPAL development. In this talk we present results of our experiments on temperature measurements in the gain medium of operating Cs DPAL at different pump power levels in the range from lasing threshold to the levels causing damage of the alkali cell windows. For precise contactless in situ temperature measurements, we used an interferometric technique, developed in our lab2 . In these experiments we demonstrated that damage of the lasing alkali cell starts in the bulk with thermal breakdown of the hydrocarbon buffer gas. The degradation processes start at definite critical temperatures of the gain medium, different for each mixture of buffer gas. At this critical temperature, the hydrocarbon and the excited alkali metal begin to react producing the characteristic black soot and, possibly, some other chemical compounds, which both harm the laser performance and significantly increase the harmful heat deposition within the laser medium. This soot, being highly absorptive, is catastrophically heated to very high temperatures that visually observed as bulk burning. This process quickly spreads to the cell windows and causes their damage. As a result, the whole cell is also contaminated with products of chemical reactions.

  8. Damage coefficients in low resistivity silicon. [solar cells

    NASA Technical Reports Server (NTRS)

    Srour, J. R.; Othmer, S.; Chiu, K. Y.; Curtis, O. L., Jr.

    1975-01-01

    Electron and proton damage coefficients are determined for low resistivity silicon based on minority-carrier lifetime measurements on bulk material and diffusion length measurements on solar cells. Irradiations were performed on bulk samples and cells fabricated from four types of boron-doped 0.1 ohm-cm silicon ingots, including the four possible combinations of high and low oxygen content and high and low dislocation density. Measurements were also made on higher resistivity boron-doped bulk samples and solar cells. Major observations and conclusions from the investigation are discussed.

  9. Tangeretin sensitizes SGS1-deficient cells by inducing DNA damage.

    PubMed

    Chong, Shin Yen; Wu, Meng-Ying; Lo, Yi-Chen

    2013-07-03

    Tangeretin, a polymethoxyflavone found in citrus peel, has been shown to have antiatherogenic, anti-inflammatory, and anticarcinogenic properties. However, the underlying target pathways are not fully characterized. We investigated the tangeretin sensitivity of yeast (Saccharomyces cerevisiae) mutants for DNA damage response or repair pathways. We found that tangeretin treatment significantly reduced (p < 0.05) survival rate, induced preferential G1 phase accumulation, and elevated the DNA double-strand break (DSB) signal γH2A in DNA repair-defective sgs1Δ cells, but had no obvious effects on wild-type cells or mutants of the DNA damage checkpoint (including tel1Δ, sml1Δ mec1Δ, sml1Δ mec1Δ tel1Δ, and rad9Δ mutants). Additionally, microarray data indicated that tangeretin treatment up-regulates genes involved in nutritional processing and down-regulates genes related to RNA processing in sgs1Δ mutants. These results suggest tangeretin may sensitize SGS1-deficient cells by increasing a marker of DNA damage and by inducing G1 arrest and possibly metabolic stress. Thus, tangeretin may be suitable for chemosensitization of cancer cells lacking DSB-repair ability.

  10. A new hand-held microfluidic cytometer for evaluating irradiation damage by analysis of the damaged cells distribution

    PubMed Central

    Wang, Junsheng; Fan, Zhiqiang; Zhao, Yile; Song, Younan; Chu, Hui; Song, Wendong; Song, Yongxin; Pan, Xinxiang; Sun, Yeqing; Li, Dongqing

    2016-01-01

    Space radiation brings uneven damages to cells. The detection of the distribution of cell damage plays a very important role in radiation medicine and the related research. In this paper, a new hand-held microfluidic flow cytometer was developed to evaluate the degree of radiation damage of cells. The device we propose overcomes the shortcomings (e.g., large volume and high cost) of commercial flow cytometers and can evaluate the radiation damage of cells accurately and quickly with potential for onsite applications. The distribution of radiation-damaged cells is analyzed by a simultaneous detection of immunofluorescence intensity of γ-H2AX and resistance pulse sensor (RPS) signal. The γ-H2AX fluorescence intensity provides information of the degree of radiation damage in cells. The ratio of the number of cells with γ-H2AX fluorescence signals to the total numbers of cells detected by RPS indicates the percentage of the cells that are damaged by radiation. The comparison experiment between the developed hand-held microfluidic flow cytometer and a commercial confocal microscope indicates a consistent and comparable detection performance. PMID:26983800

  11. Melanin photosensitizes ultraviolet light (UVC) DNA damage in pigmented cells

    SciTech Connect

    Huselton, C.A.; Hill, H.Z.

    1990-01-01

    Melanins, pigments of photoprotection and camouflage, are very photoreactive and can both absorb and emit active oxygen species. Nevertheless, black skinned individuals rarely develop skin cancer and melanin is assumed to act as a solar screen. Since DNA is the target for solar carcinogenesis, the effect of melanin on Ultraviolet (UV)-induced thymine lesions was examined in mouse melanoma and carcinoma cells that varied in melanin content. Cells prelabeled with 14C-dThd were irradiated with UVC; DNA was isolated, purified, degraded to bases by acid hydrolysis and analyzed by HPLC. Thymine dimers were detected in all of the extracts of irradiated cells.more » Melanotic and hypomelanotic but not mammary carcinoma cell DNA from irradiated cells contained hydrophilic thymine derivatives. The quantity of these damaged bases was a function of both the UVC dose and the cellular melanin content. One such derivative was identified by gas chromatography-mass spectroscopy as thymine glycol. The other appears to be derived from thymine glycol by further oxidation during acid hydrolysis of the DNA. The finding of oxidative DNA damage in melanin-containing cells suggests that melanin may be implicated in the etiology of caucasian skin cancer, particularly melanoma. Furthermore, the projected decrease in stratospheric ozone could impact in an unanticipated deleterious manner on dark-skinned individuals.« less

  12. Zingerone protects keratinocyte stem cells from UVB-induced damage.

    PubMed

    Lee, Jienny; Oh, Sae Woong; Shin, Seoung Woo; Lee, Kyung-Woo; Cho, Jae-Youl; Lee, Jongsung

    2018-01-05

    The epidermis, the outermost layer of the skin, is a stratified epithelium that protects the body from the external environment. Keratinocyte stem cells (KSCs) are involved in epidermis homeostasis by maintaining epidermal integrity through a process of constant regeneration. Ultraviolet B (UVB) radiation is a major inducer of cellular damage in the epidermis. In this study, we investigated the effects of zingerone (a phenolic compound derived from spices) on UVB-induced cellular damage in KSCs. We found that zingerone significantly inhibited cellular senescence of KSCs in response to UVB irradiation. These effects were confirmed by the senescence-associated β-galactosidase and comet assays. Zingerone decreased the production of proinflammatory cytokines such as tumor necrosis factor-α (TNF-α), interleukin-1β (IL-1β), and interleukin-6 (IL-6) in UVB-irradiated KSCs. Moreover, UVB-induced expression of p21, a cell cycle arrest-related gene, was reduced by zingerone treatment, whereas zingerone upregulated the expression of proliferation-related genes such as proliferating cell nuclear antigen (PCNA) and vascular endothelial growth factor (VEGF), in addition to anti-senescence-related genes including telomerase reverse transcriptase (TERT), histone deacetylase 1 (HDAC1), and DNA (cytosine-5)-methyltransferase 1 (DNMT1). The UVB-protective effects of zingerone were mediated by inhibition of p42/44 MAPK and p38 MAPK. Therefore, zingerone could potentially be used to protect the epidermis from UVB-induced damage. Copyright © 2017 Elsevier B.V. All rights reserved.

  13. Autophagy protects auditory hair cells against neomycin-induced damage

    PubMed Central

    He, Zuhong; Guo, Lingna; Shu, Yilai; Fang, Qiaojun; Zhou, Han; Liu, Yongze; Liu, Dingding; Lu, Ling; Zhang, Xiaoli; Ding, Xiaoqiong; Liu, Dong; Tang, Mingliang; Kong, Weijia; Sha, Suhua; Li, Huawei; Gao, Xia; Chai, Renjie

    2017-01-01

    ABSTRACT Aminoglycosides are toxic to sensory hair cells (HCs). Macroautophagy/autophagy is an essential and highly conserved self-digestion pathway that plays important roles in the maintenance of cellular function and viability under stress. However, the role of autophagy in aminoglycoside-induced HC injury is unknown. Here, we first found that autophagy activity was significantly increased, including enhanced autophagosome-lysosome fusion, in both cochlear HCs and HEI-OC-1 cells after neomycin or gentamicin injury, suggesting that autophagy might be correlated with aminoglycoside-induced cell death. We then used rapamycin, an autophagy activator, to increase the autophagy activity and found that the ROS levels, apoptosis, and cell death were significantly decreased after neomycin or gentamicin injury. In contrast, treatment with the autophagy inhibitor 3-methyladenine (3-MA) or knockdown of autophagy-related (ATG) proteins resulted in reduced autophagy activity and significantly increased ROS levels, apoptosis, and cell death after neomycin or gentamicin injury. Finally, after neomycin injury, the antioxidant N-acetylcysteine could successfully prevent the increased apoptosis and HC loss induced by 3-MA treatment or ATG knockdown, suggesting that autophagy protects against neomycin-induced HC damage by inhibiting oxidative stress. We also found that the dysfunctional mitochondria were not eliminated by selective autophagy (mitophagy) in HEI-OC-1 cells after neomycin treatment, suggesting that autophagy might not directly target the damaged mitochondria for degradation. This study demonstrates that moderate ROS levels can promote autophagy to recycle damaged cellular constituents and maintain cellular homeostasis, while the induction of autophagy can inhibit apoptosis and protect the HCs by suppressing ROS accumulation after aminoglycoside injury. PMID:28968134

  14. Modeling Microcapsule-Substrate Interactions: Repairing Damages Surfaces and Separating Damaged Cells.

    NASA Astrophysics Data System (ADS)

    Balazs, Anna

    2007-03-01

    We model two different scenarios that involve capturing the behavior of macromolecular assemblies. In the first study, we model the rolling motion of a fluid-driven, particle-filled microcapsule along a heterogeneous, adhesive substrate to determine how the release of the encapsulated nanoparticles can be harnessed to repair damage on the underlying surface. We integrate the lattice Boltzmann model for hydrodynamics and the lattice spring model for the micromechanics of elastic solids to capture the interactions between the elastic shell of the microcapsule and the surrounding fluids. A Brownian dynamics model is used to simulate the release of nanoparticles from the capsule and their diffusion into the surrounding solution. We focus on a substrate that contains a damaged region (e.g., a crack or eroded surface coating), which prevents the otherwise mobile capsule from rolling along the surface. We isolate conditions where nanoparticles released from the arrested capsule can repair the damage and thereby enable the capsules to again move along the substrate. Through these studies, we establish guidelines for designing particle-filled microcapsules that perform a ``repair and go'' function and thus, can be utilized to repair damage in microchannels and microfluidic devices. In the second study, we extend the above model of fluid-filled, elastic spheres rolling on substrates to three dimensions and thereby demonstrate a useful method for separating cells or microcapules by their compliance. In particular, we examine the fluid-driven motion of these capsules over a hard adhesive surface that contains soft stripes or a weakly adhesive surface that contains ``sticky'' stripes. As a result of their inherently different interactions with the heterogeneous substrate, particles with dissimilar stiffness are dispersed to distinct lateral locations on the surface. Since mechanically and chemically patterned surfaces can be readily fabricated through soft lithography and can

  15. Polyphosphate is a key factor for cell survival after DNA damage in eukaryotic cells.

    PubMed

    Bru, Samuel; Samper-Martín, Bàrbara; Quandt, Eva; Hernández-Ortega, Sara; Martínez-Laínez, Joan M; Garí, Eloi; Rafel, Marta; Torres-Torronteras, Javier; Martí, Ramón; Ribeiro, Mariana P C; Jiménez, Javier; Clotet, Josep

    2017-09-01

    Cells require extra amounts of dNTPs to repair DNA after damage. Polyphosphate (polyP) is an evolutionary conserved linear polymer of up to several hundred inorganic phosphate (Pi) residues that is involved in many functions, including Pi storage. In the present article, we report on findings demonstrating that polyP functions as a source of Pi when required to sustain the dNTP increment essential for DNA repair after damage. We show that mutant yeast cells without polyP produce less dNTPs upon DNA damage and that their survival is compromised. In contrast, when polyP levels are ectopically increased, yeast cells become more resistant to DNA damage. More importantly, we show that when polyP is reduced in HEK293 mammalian cell line cells and in human dermal primary fibroblasts (HDFa), these cells become more sensitive to DNA damage, suggesting that the protective role of polyP against DNA damage is evolutionary conserved. In conclusion, we present polyP as a molecule involved in resistance to DNA damage and suggest that polyP may be a putative target for new approaches in cancer treatment or prevention. Copyright © 2017 Elsevier B.V. All rights reserved.

  16. Galectin-9 ameliorates Con A-induced hepatitis by inducing CD4(+)CD25(low/int) effector T-Cell apoptosis and increasing regulatory T cell number.

    PubMed

    Lv, Kun; Zhang, Yingying; Zhang, Mengying; Zhong, Min; Suo, Qifeng

    2012-01-01

    T cell-mediated liver damage is a key event in the pathogenesis of many chronic human liver diseases, such as liver transplant rejection, primary biliary cirrhosis, and sclerosing cholangitis. We and other groups have previously reported that galectin-9, one of the β-galactoside binding animal lectins, might be potentially useful in the treatment of T cell-mediated diseases. To evaluate the direct effect of galectin-9 on hepatitis induced by concanavalin A (Con A) administration in mice and to clarify the mechanisms involved, we administered galectin-9 into mice, and evaluated its therapeutic effect on Con A-induced hepatitis. Galectin-9 was administrated i.v. to Balb/c mice 30 min before Con A injection. Compared with no treatment, galectin-9 pretreatment significantly reduced serum ALT and AST levels and improved liver histopathology, suggesting an ameliorated hepatitis. This therapeutic effect was not only attributable to a blunted Th1 immune response, but also to an increased number in regulatory T cells, as reflected in a significantly increased apoptosis of CD4(+)CD25(low/int) effector T cells and in reduced proinflammatory cytokine levels. Our findings constitute the first preclinical data indicating that interfering with TIM-3/galectin-9 signaling in vivo could ameliorate Con A-induced hepatitis. This strategy may represent a new therapeutic approach in treating human diseases involving T cell activation.

  17. Cancer cells recovering from damage exhibit mitochondrial restructuring and increased aerobic glycolysis

    SciTech Connect

    Akakura, Shin; Ostrakhovitch, Elena; Sanokawa-Akakura, Reiko

    2014-06-13

    Highlights: • Some cancer cells recover from severe damage that causes cell death in majority of cells. • Damage-Recovered (DR) cancer cells show reduced mitochondria, mDNA and mitochondrial enzymes. • DR cells show increased aerobic glycolysis, ATP, cell proliferation, and resistance to damage. • DR cells recovered from in vivo damage also show increased glycolysis and proliferation rate. - Abstract: Instead of relying on mitochondrial oxidative phosphorylation, most cancer cells rely heavily on aerobic glycolysis, a phenomenon termed as “the Warburg effect”. We considered that this effect is a direct consequence of damage which persists in cancer cells that recovermore » from damage. To this end, we studied glycolysis and rate of cell proliferation in cancer cells that recovered from severe damage. We show that in vitro Damage-Recovered (DR) cells exhibit mitochondrial structural remodeling, display Warburg effect, and show increased in vitro and in vivo proliferation and tolerance to damage. To test whether cancer cells derived from tumor microenvironment can show similar properties, we isolated Damage-Recovered (T{sup DR}) cells from tumors. We demonstrate that T{sup DR} cells also show increased aerobic glycolysis and a high proliferation rate. These findings show that Warburg effect and its consequences are induced in cancer cells that survive severe damage.« less

  18. Frequent retinal ganglion cell damage after acute optic neuritis.

    PubMed

    Brandt, Alexander U; Specovius, Svenja; Oberwahrenbrock, Timm; Zimmermann, Hanna G; Paul, Friedemann; Costello, Fiona

    2018-04-09

    To identify the extent of ganglion cell damage after first-time optic neuritis (ON) using the inter-ocular difference between affected and fellow eyes, and whether this approach is able to detect more patients suffering from ganglion cell damage than using absolute values. Thirty-four patients with first-time unilateral ON were followed for a median 413 days. Patients underwent optical coherence tomography testing to determine ganglion cell plus inner plexiform layer thickness (GCIP). Ganglion cell loss was quantified as GCIP difference between ON-affected and fellow eyes (inter-GCIP) and was compared against measurements from 93 healthy controls (HC). Visual function was assessed with high contrast visual acuity; and standard automated perimetry-derived measures of mean deviation and foveal threshold. At clinical presentation after median 19 days from symptom onset, 47.1% of patients showed early GCIP thinning in the ON-affected eye based on inter-GCIP. At the last visit acute ON was associated with 16.1 ± 10.0 µm GCIP thinning compared to fellow eyes (p = 3.669e-06). Based on inter-GCIP, 84.9% of ON patients sustained GCIP thinning in their affected eye at the last visit, whereas using absolute values only 71.0% of patients suffered from GCIP thinning (p = 0.002076). Only 32.3% of these patients had abnormal visual function. The best predictor of GCIP thinning as a measure of ON severity at the last visit was worse visual field mean deviation at clinical presentation. Inter-ocular GCIP identifies significantly more eyes suffering damage from ON than absolute GCIP, visual fields or visual acuity loss. Effective interventional options are needed to prevent ganglion cell loss. Copyright © 2018. Published by Elsevier B.V.

  19. DNA damage response induced by HZE particles in human cells

    NASA Astrophysics Data System (ADS)

    Chen, David; Aroumougame, Asaithamby

    Convincing evidences indicate that high-linear energy transfer (LET) ionizing radiation (IR) induced complex DNA lesions are more difficult to repair than isolated DNA lesions induced by low-LET IR; this has been associated with the increased RBE for cell killing, chromosomal aberrations, mutagenesis, and carcinogenesis in high energy charged-particle irradiated human cells. We have employed an in situ method to directly monitor induction and repair of clustered DNA lesions at the single-cell level. We showed, consistent with biophysical modeling, that the kinetics of loss of clustered DNA lesions was substantially compromised in human fibroblasts. The unique spatial distribution of different types of DNA lesions within the clustered damages determined the cellular ability to repair these damages. Importantly, examination of metaphase cells derived from HZE particle irradiated cells revealed that the extent of chromosome aberrations directly correlated with the levels of unrepaired clustered DNA lesions. In addition, we used a novel organotypic human lung three-dimensional (3D) model to investigate the biological significance of unrepaired DNA lesions in differentiated lung epithelial cells. We found that complex DNA lesions induced by HZE particles were even more difficult to be repaired in organotypic 3D culture, resulting enhanced cell killing and chromosome aberrations. Our data suggest that DNA repair capability in differentiated cells renders them vulnerable to DSBs, promoting genome instability that may lead to carcinogenesis. As the organotypic 3D model mimics human lung, it opens up new experimental approaches to explore the effect of radiation in vivo and will have important implications for evaluating radiation risk in human tissues.

  20. FOXO transcription factors enforce cell cycle checkpoints and promote survival of hematopoietic cells after DNA damage.

    PubMed

    Lei, Hong; Quelle, Frederick W

    2009-08-01

    The PI3K/AKT signaling pathway contributes to cell cycle progression of cytokine-dependent hematopoietic cells under normal conditions, and it is absolutely required to override DNA damage-induced cell cycle arrest checkpoints in these cells. Phosphatidylinositol-3-kinase (PI3K)/AKT activity also correlates with Cdk2 activity in hematopoietic cells, suggesting that Cdk2 activation may be a relevant end point for this signaling pathway. However, mediators downstream of AKT in this pathway have not been defined. The forkhead transcription factor O (FOXO) family are negatively regulated by AKT-dependent phosphorylation and are known regulators of genes affecting cell cycle progression. We show that enhanced FOXO activity replicates the effect of PI3K inhibitors in enforcing G(1) and G(2) phase arrest after DNA damage. Conversely, knockdown of endogenous FOXO proteins increased Cdk2 activity and overrode DNA damage checkpoints in cells lacking PI3K activity. Moreover, loss of FOXO activity caused an increase in sensitivity to cisplatin-induced cell death, which was associated with failure to arrest cell cycle progression in the face of DNA damage caused by this chemotherapeutic agent. These cell cycle arrests were dependent on p27 expression when mediated by FOXO3a alone, but also involve p27-independent mechanisms when promoted by endogenous FOXO proteins. Together, these observations show that FOXO proteins enforce DNA damage-induced cell cycle arrest in hematopoietic cells. Inhibition of FOXO activity by cytokine-induced PI3K/AKT signaling is sufficient to override these DNA damage-induced cell cycle checkpoints, but may negatively impact hematopoietic cell viability.

  1. Simulation of ion induced radiation damage in cells

    NASA Astrophysics Data System (ADS)

    Friedland, W.; Jacob, P.; Paretzke, H. G.; Ottolenghi, A.; Ballarini, F.; Dingfelder, M.

    The biophysical simulation code PARTRAC has been used in several studies of DNA damage induced by various radiation qualities including photons electrons protons alphas and ions heavier than alpha particles Ion-electron interaction cross sections are taken from isotachic protons scaled by Z eff 2 with the effective charge calculated according to the Barkas formula Recently ion type dependent angular distributions were introduced for intermediate secondary electron energies taking into account the different kinematic scaling of the constituents of the electron spectra Calculated stopping powers radial dose distributions and secondary electron spectra were found in good agreement with available experimental and theoretical results Radiation damage to DNA is determined in PARTRAC by superposition of the calculated track structures with a DNA target model taking into account direct effects from coincidences of ionisations and atoms within the DNA helix as well as indirect effects due to interactions of OH radicals produced in water surrounding the DNA For a simulation of radiation effects in human cells this target model comprises several genomic structure levels from the DNA double-helix up to chromosomes Calculated DNA damage due to irradiation of human fibroblast cells by ions of boron nitrogen and neon was compared to corresponding experimental data The calculated total yield of DSB per dose showed saturation behaviour with an RBE of about 2 whereas experimental data had a decreasing tendency with increasing LET to RBE values

  2. Electrochemically reduced water protects neural cells from oxidative damage.

    PubMed

    Kashiwagi, Taichi; Yan, Hanxu; Hamasaki, Takeki; Kinjo, Tomoya; Nakamichi, Noboru; Teruya, Kiichiro; Kabayama, Shigeru; Shirahata, Sanetaka

    2014-01-01

    Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50-200 μM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 μM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca(2+) influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW.

  3. Electrochemically Reduced Water Protects Neural Cells from Oxidative Damage

    PubMed Central

    Hamasaki, Takeki; Kinjo, Tomoya; Nakamichi, Noboru; Teruya, Kiichiro; Kabayama, Shigeru

    2014-01-01

    Aging-related neurodegenerative disorders are closely associated with mitochondrial dysfunction and oxidative stresses and their incidence tends to increase with aging. Brain is the most vulnerable to reactive species generated by a higher rate of oxygen consumption and glucose utilization compared to other organs. Electrochemically reduced water (ERW) was demonstrated to scavenge reactive oxygen species (ROS) in several cell types. In the present study, the protective effect of ERW against hydrogen peroxide (H2O2) and nitric oxide (NO) was investigated in several rodent neuronal cell lines and primary cells. ERW was found to significantly suppress H2O2 (50–200 μM) induced PC12 and SFME cell deaths. ERW scavenged intracellular ROS and exhibited a protective effect against neuronal network damage caused by 200 μM H2O2 in N1E-115 cells. ERW significantly suppressed NO-induced cytotoxicity in PC12 cells despite the fact that it did not have the ability to scavenge intracellular NO. ERW significantly suppressed both glutamate induced Ca2+ influx and the resulting cytotoxicity in primary cells. These results collectively demonstrated for the first time that ERW protects several types of neuronal cells by scavenging ROS because of the presence of hydrogen and platinum nanoparticles dissolved in ERW. PMID:25383141

  4. Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.

    PubMed

    Cui, Xiaofeng; Dean, Delphine; Ruggeri, Zaverio M; Boland, Thomas

    2010-08-15

    Thermal inkjet printing technology has been applied successfully to cell printing. However, there are concerns that printing process may cause cell damages or death. We conducted a comprehensive study of thermal inkjet printed Chinese hamster ovary (CHO) cells by evaluating cell viability and apoptosis, and possible cell membrane damages. Additionally, we studied the cell concentration of bio-ink and found optimum printing of concentrations around 8 million cells per mL. Printed cell viability was 89% and only 3.5% apoptotic cells were observed after printing. Transient pores were developed in the cell membrane of printed cells. Cells were able to repair these pores within 2 h after printing. Green fluorescent protein (GFP) DNA plasmids were delivered to CHO-S cells by co-printing. The transfection efficiency is above 30%. We conclude that thermal inkjet printing technology can be used for precise cell seeding with minor effects and damages to the printed mammalian cells. The printing process causes transient pores in cell membranes, a process which has promising applications for gene and macroparticles delivery to induce the biocompatibility or growth of engineered tissues.

  5. Ecabet sodium alleviates neomycin-induced hair cell damage.

    PubMed

    Rah, Yoon Chan; Choi, June; Yoo, Myung Hoon; Yum, Gunhwee; Park, Saemi; Oh, Kyoung Ho; Lee, Seung Hoon; Kwon, Soon Young; Cho, Seung Hyun; Kim, Suhyun; Park, Hae-Chul

    2015-12-01

    Ecabet sodium (ES) is currently applied to some clinical gastrointestinal disease primarily by the inhibition of the ROS production. In this study, the protective role of ES was evaluated against the neomycin-induced hair cell loss using zebrafish experimental animal model. Zebrafish larvae (5-7 dpf), were treated with each of the following concentrations of ES: 5, 10, 20, 40, and 80 μg/mL for 1 h, followed by 125 μM neomycin for 1h. The positive control group was established by 125 μM neomycin-only treatment (1h) and the negative control group with no additional chemicals was also established. Hair cells inside four neuromasts ( SO1, SO2, O1, OC1) were assessed using fluorescence microscopy (n = 10). Hair cell survival was calculated as the mean number of viable hair cells for each group. Apoptosis and mitochondrial damage were investigated using special staining (TUNEL and DASPEI assay, respectively), and compared among groups. Ultrastructural changes were evaluated using scanning electron microscopy. Pre-treatment group with ES increased the mean number of viable hair cells as a dose-dependent manner achieving almost same number of viable hair cells with 40 μM/ml ES treatment (12.98 ± 2.59 cells) comparing to that of the negative control group (14.15 ± 1.39 cells, p = 0.72) and significantly more number of viable hair cells than that of the positive control group (7.45 ± 0.91 cells, p < 0.01). The production of reactive oxygen species significantly increased by 183% with 125 μM neomycin treatment than the negative control group and significantly decreased down to 105% with the pre-treatment with 40 μM/ml ES (n = 40, p = 0.04). A significantly less number of TUNEL-positive cells (reflecting apoptosis, p < 0.01) and a significantly increased DASPEI reactivity (reflecting viable mitochondria, p < 0.01) were observed in 40 μM/ml ES pre-treatment group. Our data suggest that ES could protect against neomycin-induced hair cell loss possibly by reducing

  6. Burst annealing of electron damage in silicon solar cells

    NASA Technical Reports Server (NTRS)

    Day, A. C.; Horne, W. E.; Thompson, M. A.; Lancaster, C. A.

    1985-01-01

    A study has been performed of burst annealing of electron damage in silicon solar cells. Three groups of cells consisting of 3 and 0.3 ohm-cm silicon were exposed to fluences of 2 x 10 to the 14th power, 4 x 10 to the 14th power, and 8 x 10 to the 14th power 1-MeV electrons/sq cm, respectively. They were subsequently subjected to 1-minute bursts of annealing at 500 C. The 3 ohm-cm cells showed complete recovery from each fluence level. The 0.3 ohm-cm cells showed complete recovery from the 2 x 10 to the 14th power e/sq cm fluence; however, some of the 0.3 ohm-cm cells did not recover completely from the higher influences. From an analysis of the results it is concluded that burst annealing of moderate to high resistivity silicon cell arrays in space is feasible and that with more complete understanding, even the potentially higher efficiency low resistivity cells may be usable in annealable arrays in space.

  7. Deoxyactein Isolated from Cimicifuga racemosa protects osteoblastic MC3T3-E1 cells against antimycin A-induced cytotoxicity.

    PubMed

    Choi, Eun Mi

    2013-06-01

    Deoxyactein is one of the major constituents isolated from Cimicifuga racemosa. In the present study, we investigated the protective effects of deoxyactein on antimycin A (mitochondrial electron transport inhibitor)-induced toxicity in osteoblastic MC3T3-E1 cells. Exposure of MC3T3-E1 cells to antimycin A caused significant cell viability loss, as well as mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, intracellular calcium ([Ca(2+) ]i ) elevation and oxidative stress. Pretreatment with deoxyactein prior to antimycin A exposure significantly reduced antimycin A-induced cell damage by preventing mitochondrial membrane potential dissipation, complex IV inactivation, ATP loss, [Ca(2+) ]i elevation and oxidative stress. Moreover, deoxyactein increased the activation of PI3K (phosphoinositide 3-kinase), Akt (protein kinase B) and CREB (cAMP-response element-binding protein) inhibited by antimycin A. All these data indicate that deoxyactein may reduce or prevent osteoblasts degeneration in osteoporosis or other degenerative disorders. Copyright © 2011 John Wiley & Sons, Ltd.

  8. DNA damage in embryonic stem cells caused by nanodiamonds.

    PubMed

    Xing, Yun; Xiong, Wei; Zhu, Lin; Osawa, Eiji; Hussin, Saber; Dai, Liming

    2011-03-22

    Because of their unique photoluminescence and magnetic properties, nanodiamonds (NDs) are promising for biomedical imaging and therapeutical applications. However, these biomedical applications will hardly be realized unless the potential hazards of NDs to humans and other biological systems are ascertained. Previous studies performed in our group and others have demonstrated the excellent biocompatibility of NDs in a variety of cell lines without noticeable cytotoxicity. In the present paper, we report the first genotoxicity study on NDs. Our results showed that incubation of embryonic stem cells with NDs led to slightly increased expression of DNA repair proteins, such as p53 and MOGG-1. Oxidized nanodiamonds (O-NDs) were demonstrated to cause more DNA damage than the pristine/raw NDs (R-NDs), showing the surface chemistry specific genotoxicity. However, the DNA damages caused by either the O-NDs or the R-NDs are much less severe than those caused by multiwalled carbon nanotubes (MWNTs) observed in our previous study. These findings should have important implications for future applications of NDs in biological applications.

  9. Photodynamic damage of glial cells in crayfish ventral nerve cord

    NASA Astrophysics Data System (ADS)

    Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

    2011-03-01

    Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

  10. Photodynamic damage of glial cells in crayfish ventral nerve cord

    NASA Astrophysics Data System (ADS)

    Kolosov, M. S.; Duz, E.; Uzdensky, A. B.

    2010-10-01

    Photodynamic therapy (PDT) is a promising method for treatment of brain tumors, the most of which are of glial origin. In the present work we studied PDT-mediated injury of glial cells in nerve tissue, specifically, in abdominal connectives in the crayfish ventral nerve cord. The preparation was photosensitized with alumophthalocyanine Photosens and irradiated 30 min with the diode laser (670 nm, 0.1 or 0.15 W/cm2). After following incubation in the darkness during 1- 10 hours it was fluorochromed with Hoechst 33342 and propidium iodide to reveal nuclei of living, necrotic and apoptotic cells. The chain-like location of the glial nuclei allowed visualization of those enveloping giant axons and blood vessels. The level of glial necrosis in control preparations was about 2-5 %. Apoptosis was not observed in control preparations. PDT significantly increased necrosis of glial cells to 52 or 67 % just after irradiation with 0.1 or 0.15 W/cm2, respectively. Apoptosis of glial cells was observed only at 10 hours after light exposure. Upper layers of the glial envelope of the connectives were injured stronger comparing to deep ones: the level of glial necrosis decreased from 100 to 30 % upon moving from the connective surface to the plane of the giant axon inside the connective. Survival of glial cells was also high in the vicinity of blood vessels. One can suggest that giant axons and blood vessels protect neighboring glial cells from photodynamic damage. The mechanism of such protective action remains to be elucidated.

  11. Reconstitution of the cellular response to DNA damage in vitro using damage-activated extracts from mammalian cells

    SciTech Connect

    Roper, Katherine; Coverley, Dawn, E-mail: dc17@york.ac.uk

    2012-03-10

    In proliferating mammalian cells, DNA damage is detected by sensors that elicit a cellular response which arrests the cell cycle and repairs the damage. As part of the DNA damage response, DNA replication is inhibited and, within seconds, histone H2AX is phosphorylated. Here we describe a cell-free system that reconstitutes the cellular response to DNA double strand breaks using damage-activated cell extracts and naieve nuclei. Using this system the effect of damage signalling on nuclei that do not contain DNA lesions can be studied, thereby uncoupling signalling and repair. Soluble extracts from G1/S phase cells that were treated with etoposidemore » before isolation, or pre-incubated with nuclei from etoposide-treated cells during an in vitro activation reaction, restrain both initiation and elongation of DNA replication in naieve nuclei. At the same time, H2AX is phosphorylated in naieve nuclei in a manner that is dependent upon the phosphatidylinositol 3-kinase-like protein kinases. Notably, phosphorylated H2AX is not focal in naieve nuclei, but is evident throughout the nucleus suggesting that in the absence of DNA lesions the signal is not amplified such that discrete foci can be detected. This system offers a novel screening approach for inhibitors of DNA damage response kinases, which we demonstrate using the inhibitors wortmannin and LY294002. -- Highlights: Black-Right-Pointing-Pointer A cell free system that reconstitutes the response to DNA damage in the absence of DNA lesions. Black-Right-Pointing-Pointer Damage-activated extracts impose the cellular response to DNA damage on naieve nuclei. Black-Right-Pointing-Pointer PIKK-dependent response impacts positively and negatively on two separate fluorescent outputs. Black-Right-Pointing-Pointer Can be used to screen for inhibitors that impact on the response to damage but not on DNA repair. Black-Right-Pointing-Pointer LY294002 and wortmannin demonstrate the system's potential as a pathway focused

  12. Effects of S-adenosylhomocysteine and homocysteine on DNA damage and cell cytotoxicity in murine hepatic and microglia cell lines.

    PubMed

    Liu, Chia-Chyuan; Ho, Wen-Yueh; Leu, Kuen-Lin; Tsai, Hsin-Mao; Yang, Tsai-Hsiu

    2009-01-01

    Limited research has been performed on S-adenosylhomocysteine (SAH) or homocysteine (Hcy)-evoked cell damage in hepatic and neuronal cells. In this study, we assessed effects of SAH or Hcy on cell cytotoxicity and DNA damage in hepatic and neuronal cells and attempted to find the underlying mechanism. Cell cytotoxicity and DNA damage were evaluated in murine hepatic cells (BNL CL.2 cell line) and microglia cells (BV-2 cell line) with SAH or Hcy treatment for 48 h. The influences of SAH or Hcy on lipid peroxidation and DNA methylation were also measured in both cell lines. SAH (5-20 microM) or Hcy (1-5 mM) dose dependently inhibited cell cytotoxicity and enhanced DNA damage in both types of cells. Furthermore, SAH treatment markedly increased intracellular SAH levels and DNA hypomethylation, whereas Hcy caused minimal effects on these two parameters at much higher concentrations. Hcy significantly induced lipid peroxidation, but not SAH. The present results show that SAH might cause cellular DNA damage in hepatic and microglia cells by DNA hypomethylation, resulting in irreversible DNA damage and increased cell cytotoxicity. In addition, higher Hcy could induce cellular DNA damage through increased lipid peroxidation and DNA hypomethylation. We suggest that SAH is a better marker of cell damage than Hcy in hepatic and microglia cells.

  13. A pharmacologically-based array to identify targets of cyclosporine A-induced toxicity in cultured renal proximal tubule cells

    SciTech Connect

    Sarró, Eduard, E-mail: eduard.sarro@vhir.org; Renal Physiopathology, CIBBIM-Nanomedicine, Vall d'Hebron Research Institute; Jacobs-Cachá, Conxita, E-mail: conxita.jacobs@vhir.org

    2012-01-15

    Mechanisms of cyclosporine A (CsA)-induced nephrotoxicity were generally thought to be hemodynamic in origin; however, there is now accumulating evidence of a direct tubular effect. Although genomic and proteomic experiments by our group and others provided overall information on genes and proteins up- or down-regulated by CsA in proximal tubule cells (PTC), a comprehensive view of events occurring after CsA exposure remains to be described. For this purpose, we applied a pharmacologic approach based on the use of known activities of a large panel of potentially protective compounds and evaluated their efficacy in preventing CsA toxicity in cultured mouse PTC.more » Our results show that compounds that blocked protein synthesis and apoptosis, together with the CK2 inhibitor DMAT and the PI3K inhibitor apigenin, were the most efficient in preventing CsA toxicity. We also identified GSK3, MMPs and PKC pathways as potential targets to prevent CsA damage. Additionally, heparinase-I and MAPK inhibitors afforded partial but significant protection. Interestingly, antioxidants and calcium metabolism-related compounds were unable to ameliorate CsA-induced cytotoxicity. Subsequent experiments allowed us to clarify the hierarchical relationship of targeted pathways after CsA treatment, with ER stress identified as an early effector of CsA toxicity, which leads to ROS generation, phenotypical changes and cell death. In summary, this work presents a novel experimental approach to characterizing cellular responses to cytotoxics while pointing to new targets to prevent CsA-induced toxicity in proximal tubule cells. Highlights: ► We used a novel pharmacological approach to elucidate cyclosporine (CsA) toxicity. ► The ability of a broad range of compounds to prevent CsA toxicity was evaluated. ► CsA toxicity was monitored using LDH release assay and PARP cleavage. ► Protein synthesis, PI3K, GSK3, MMP, PKC and caspase inhibitors prevented CsA toxicity. ► We also

  14. Evaluation of the damage of cell wall and cell membrane for various extracellular polymeric substance extractions of activated sludge.

    PubMed

    Guo, Xuesong; Liu, Junxin; Xiao, Benyi

    2014-10-20

    Extracellular polymeric substances (EPS) are susceptible to contamination by intracellular substances released during the extraction of EPS owing to the damage caused to microbial cell structures. The damage to cell walls and cell membranes in nine EPS extraction processes of activated sludge was evaluated in this study. The extraction of EPS (including proteins, carbohydrates and DNA) was the highest using the NaOH extraction method and the lowest using formaldehyde extraction. All nine EPS extraction methods in this study resulted in cell wall and membrane damage. The damage to cell walls, evaluated by 2-keto-3-deoxyoctonate (KDO) and N-acetylglucosamine content changes in extracted EPS, was the most significant in the NaOH extraction process. Formaldehyde extraction showed a similar extent of damage to cell walls to those detected in the control method (centrifugation), while those in the formaldehyde-NaOH and cation exchange resin extractions were slightly higher than those detected in the control. N-acetylglucosamine was more suitable than KDO for the evaluation of cell wall damage in the EPS extraction of activated sludge. The damage to cell membranes was characterized by two fluorochromes (propidium iodide and FITC Annexin V) with flow cytometry (FCM) measurement. The highest proportion of membrane-damaged cells was detected in NaOH extraction (26.54% of total cells) while membrane-damaged cells comprised 8.19% of total cells in the control. Copyright © 2014 Elsevier B.V. All rights reserved.

  15. Brefeldin A induces apoptosis and cell cycle blockade in glioblastoma cell lines.

    PubMed

    Pommepuy, Isabelle; Terro, Faraj; Petit, Barbara; Trimoreau, Frank; Bellet, Virginie; Robert, Sandrine; Hugon, Jacques; Labrousse, François; Yardin, Catherine

    2003-01-01

    Brefeldin A (BFA), a fungal metabolite known to affect the structure and function of the Golgi apparatus, has recently been shown to induce apoptosis and cell growth inhibition in various human cell lines. Glioblastomas (GB) are cerebral tumors with poor prognosis, which display resistance to current therapies including radio- and chemotherapy. The objective of this study was to investigate BFA effects in three human GB cell lines (SA4, SA146 and U87MG cells). Compared with control cells, about 60% of cell growth inhibition was observed in BFA (100 ng/ml for 24 h)-exposed cells in the three cell lines. Furthermore, in SA4 and SA146 cells, BFA was able to induce a time- and dose-dependent apoptosis detected by DAPI staining, TUNEL assay and flow-cytometric analysis. Since p53 expression was not modified after BFA exposure, BFA-induced apoptosis may follow a p53-independent pathway, as already reported. In the same way, BFA did not alter Bcl-2, Bax and Mcl-1 expression. Cell cycle analysis revealed a cell cycle arrest in early G0/G1 phase with an increase in G0/G1 cell population (70% in control cells vs. 83% in exposed cells) associated with a decrease in the S cell population (14% in control cells vs. 5.5% in exposed cells). The Ki67 labeling index also confirmed the cell cycle blockade. Our results suggest that BFA may be a potent cell cycle modulator and inducer of apoptosis in GB cell lines, and therefore may become a promising candidate for the chemotherapeutic treatment of gliomas. Copyright 2003 S. Karger AG, Basel

  16. Cyclosporine A induced epithelial-mesenchymal transition in human renal proximal tubular epithelial cells.

    PubMed

    McMorrow, Tara; Gaffney, Michelle M; Slattery, Craig; Campbell, Eric; Ryan, Michael P

    2005-10-01

    Tubulointerstitial fibrosis is a relatively common and sinister complication of cyclosporine A (CsA) therapy that limits its clinical use. CsA may have direct effects on renal tubular epithelial cells by promoting epithelial-mesenchymal transition (EMT). EMT plays an important role in embryonic development and tumourigenesis and has been described in organ remodelling during fibrogenesis. In this study, we investigated the effects of CsA on a human renal cell line as a model system to test the hypothesis that CsA can induce renal EMT. Human renal proximal tubular cells were treated with CsA (0.42-42 microm) for periods up to 72 h. Viability was assessed by the Alamar Blue assay. Morphological changes were assessed by phase contrast microscopy. The effects on epithelial adherens molecule, beta-catenin and stress fibre protein, F-actin were analysed by indirect immunofluorescence. Reverse transcription--polymerse chain reaction was performed to measure the mRNA levels of extracellular matrix components. Expression of transforming growth factor-beta was measured by western blotting. Expression and activity of matrix metalloproteinases were measured by gelatin zymography. CsA induced striking morphological changes in epithelial cells, including changes in cellular morphology, F-actin stress fibre formation, delocalization of the adherens junction protein beta-catenin and increased levels of collagen IV and fibronectin. In addition, CsA-induced EMT was associated with increased TGF-beta1 protein levels and EMT was markedly attenuated in the presence of anti-TGF-beta1 antibody. CsA-induced EMT was also associated with increased expression of connective tissue growth factor (CTGF) suggesting that this molecule may serve as downstream mediator of TGF-beta1 pro-fibrotic activity in this setting. In aggregate, these data suggest that CsA is a direct stimulus for EMT in renal tubule epithelial cells and implicate TGF-beta1 and CTGF as mediators of this response. The further

  17. Inhibition of exportin-1 function results in rapid cell cycle-associated DNA damage in cancer cells.

    PubMed

    Burke, Russell T; Marcus, Joshua M; Orth, James D

    2017-06-13

    Selective inhibitors of nuclear export (SINE) are small molecules in development as anti-cancer agents. The first-in-class SINE, selinexor, is in clinical trials for blood and solid cancers. Selinexor forms a covalent bond with exportin-1 at cysteine-528, and blocks its ability to export cargos. Previous work has shown strong cell cycle effects and drug-induced cell death across many different cancer-derived cell lines. Here, we report strong cell cycle-associated DNA double-stranded break formation upon the treatment of cancer cells with SINE. In multiple cell models, selinexor treatment results in the formation of clustered DNA damage foci in 30-40% of cells within 8 hours that is dependent upon cysteine-528. DNA damage strongly correlates with G1/S-phase and decreased DNA replication. Live cell microscopy reveals an association between DNA damage and cell fate. Cells that form damage in G1-phase more often die or arrest, while those damaged in S/G2-phase frequently progress to cell division. Up to half of all treated cells form damage foci, and most cells that die after being damaged, were damaged in G1-phase. By comparison, non-transformed cell lines show strong cell cycle effects but little DNA damage and less death than cancer cells. Significant drug combination effects occur when selinexor is paired with different classes of agents that either cause DNA damage or that diminish DNA damage repair. These data present a novel effect of exportin-1 inhibition and provide a strong rationale for multiple combination treatments of selinexor with agents that are currently in use for the treatment of different solid cancers.

  18. miR-151a induces partial EMT by regulating E-cadherin in NSCLC cells

    PubMed Central

    Daugaard, I; Sanders, K J; Idica, A; Vittayarukskul, K; Hamdorf, M; Krog, J D; Chow, R; Jury, D; Hansen, L L; Hager, H; Lamy, P; Choi, C L; Agalliu, D; Zisoulis, D G; Pedersen, I M

    2017-01-01

    miR-151a and its host gene, focal adhesion kinase, FAK, are located in a region of chromosome 8q that is frequently amplified in solid tumors, including lung cancer. Lung cancer is the leading cause of cancer deaths worldwide and metastasis remains the major challenge in battling lung cancer mortality. Here, we demonstrate that miR-151a is overexpressed in non-small cell lung cancer (NSCLC) patient specimens, as compared to healthy lung. In addition, miR-151a overexpression promotes proliferation, epithelial-to-mesenchymal transition (EMT) and induces tumor cell migration and invasion of NSCLC cells. Blocking miR-151a expression using anti-miR-151a approaches significantly reduced NCSLC cell proliferative and motility potential. Furthermore, we determined that miR-151a significantly regulates E-cadherin expression. Finally, functional rescue experiments determined that overexpression of E-cadherin in miR-151a NSCLC cell lines potently repressed miR-151a-induced partial EMT and cell migration of NSCLC cells. In conclusion, our findings suggest that miR-151a functions as an oncomiR in NSCLC by targeting E-cadherin mRNA and inducing proliferation, migration and partial EMT. PMID:28759022

  19. Interplay between autophagy and apoptosis in TrkA-induced cell death.

    PubMed

    Dadakhujaev, Shorafidinkhuja; Jung, Eun Joo; Noh, Hae Sook; Hah, Young-Sool; Kim, Chang Jae; Kim, Deok Ryong

    2009-01-01

    Autophagy is a self-eating process to eradicate damaged proteins or organelles in cells. This process begins with formation of a double-membrane structure, called an autophagosome, which can sequester soluble proteins and organelles eventually degraded by lysosomal proteases after fusion with the lysosome. Autophagy was initially identified as a cell survival mechanism under stress conditions such as nutrient deprivation. More recently, it is also considered as type-II programmed cell death. In our recent report, we observed that overexpression of TrkA caused massive cell death via both apoptosis and autophagy. Overexpression of TrkA abated catalase activity and subsequently resulted in the production of a large amount of reactive oxygen species in cells. These consequences led to autophagic cell death. The autophagic cell death in TrkA-overexpressing cells was validated by GFP-LC3 dot formation, production of autophagosomes or acidic vacuoles, LC3 lipidation, and depletion of autopahgy-related genes. In addition, we also observed some evidence for apoptosis in TrkA-expressing cells. Many cells expressing TrkA exhibited annexin V-positive staining, activation of caspase-7 and BAX. Moreover, TrkA activated the JNK pathway, leading to phosphorylation of H2AX. In this report, we suggest that two cell death mechanisms occur simultaneously and interlink with each other. The JNK-calpain pathway might be a central process to mediate the two processes in TrkA-overexpressing cells, although further study still remains to prove the interplay between autophagy and apoptosis.

  20. Alcoholic beverages and gastric epithelial cell viability: effect on oxidative stress-induced damage.

    PubMed

    Loguercio, C; Tuccillo, C; Federico, A; Fogliano, V; Del Vecchio Blanco, C; Romano, M

    2009-12-01

    Alcohol is known to cause damage to the gastric epithelium independently of gastric acid secretion. Different alcoholic beverages exert different damaging effects in the stomach. However, this has not been systematically evaluated. Moreover, it is not known whether the non-alcoholic components of alcoholic beverages also play a role in the pathogenesis of gastric epithelial cell damage. Therefore, this study was designed to evaluate whether different alcoholic beverages, at a similar ethanol concentration, exerted different damaging effect in gastric epithelial cells in vitro. Moreover, we evaluated whether pre-treatment of gastric epithelial cells with alcoholic beverages prevented oxidative stress-induced damage to gastric cells. Cell damage was assessed, in MKN-28 gastric epithelial cells, by MTT assay. Oxidative stress was induced by incubating cells with xanthine and xanthine oxidase. Gastric cell viability was assessed following 30, 60, and 120 minutes incubation with ethanol 17.5-125 mg/ml(-1) or different alcoholic beverages (i.e., beer, white wine, red wine, spirits) at comparable ethanol concentration. Finally, we assessed whether pre-incubation with red wine (with or without ethanol) prevented oxidative stress-induced cell damage. Red wine caused less damage to gastric epithelial cells in vitro compared with other alcoholic beverages at comparable ethanol concentration. Pre-treatment with red wine, but not with dealcoholate red wine, significantly and time-dependently prevented oxidative stress-induced cell damage. 1) red wine is less harmful to gastric epithelial cells than other alcoholic beverages; 2) this seems related to the non-alcoholic components of red wine, because other alcoholic beverages with comparable ethanol concentration exerted more damage than red wine; 3) red wine prevents oxidative stress-induced cell damage and this seems to be related to its ethanol content.

  1. A biophysical model of cell evolution after cytotoxic treatments: Damage, repair and cell response.

    PubMed

    Tomezak, M; Abbadie, C; Lartigau, E; Cleri, F

    2016-01-21

    We present a theoretical agent-based model of cell evolution under the action of cytotoxic treatments, such as radiotherapy or chemotherapy. The major features of cell cycle and proliferation, cell damage and repair, and chemical diffusion are included. Cell evolution is based on a discrete Markov chain, with cells stepping along a sequence of discrete internal states from 'normal' to 'inactive'. Probabilistic laws are introduced for each type of event a cell can undergo during its life: duplication, arrest, senescence, damage, reparation, or death. We adjust the model parameters on a series of cell irradiation experiments, carried out in a clinical LINAC, in which the damage and repair kinetics of single- and double-strand breaks are followed. Two showcase applications of the model are then presented. In the first one, we reconstruct the cell survival curves from a number of published low- and high-dose irradiation experiments. We reobtain a very good description of the data without assuming the well-known linear-quadratic model, but instead including a variable DSB repair probability. The repair capability of the model spontaneously saturates to an exponential decay at increasingly high doses. As a second test, we attempt to simulate the two extreme possibilities of the so-called 'bystander' effect in radiotherapy: the 'local' effect versus a 'global' effect, respectively activated by the short-range or long-range diffusion of some factor, presumably secreted by the irradiated cells. Even with an oversimplified simulation, we could demonstrate a sizeable difference in the proliferation rate of non-irradiated cells, the proliferation acceleration being much larger for the global than the local effect, for relatively small fractions of irradiated cells in the colony. Copyright © 2015 Elsevier Ltd. All rights reserved.

  2. Cardiac cell sheet transplantation improves damaged heart function via superior cell survival in comparison with dissociated cell injection.

    PubMed

    Sekine, Hidekazu; Shimizu, Tatsuya; Dobashi, Izumi; Matsuura, Katsuhisa; Hagiwara, Nobuhisa; Takahashi, Masafumi; Kobayashi, Eiji; Yamato, Masayuki; Okano, Teruo

    2011-12-01

    Regenerative therapies have currently emerged as one of the most promising treatments for repair of the damaged heart. Recently, numerous researchers reported that isolated cell injection treatments can improve heart function in myocardial infarction models. However, significant cell loss due to primary hypoxia or cell wash-out and difficulty to control the location of the grafted cells remains problem. As an attempt to overcome these limitations, we have proposed cell sheet-based tissue engineering, which involves stacking confluently cultured cells (two-dimensional), cell sheets, to construct three-dimensional cell-dense tissues. Cell sheet transplantation has been able to recover damaged heart function. However, no detailed analysis for transplanted cell survival has been previously performed. The present study compared the survival of cardiac cell sheet transplantation to direct cell injection in a rat myocardial infarction model. Luciferase-expressing neonatal rat cardiac cells were harvested as cell sheets from temperature-responsive culture dishes. The transplantation of cell sheets was compared to the direct injection of isolated cells dissociated with trypsin-ethylenediaminetetraacetic acid. These grafts were transplanted to infarcted rat hearts and cardiac function was assessed by echocardiography at 2 and 4 weeks after transplantation. In vivo bioluminescence and histological analyses were performed to examine cell survival. Cell sheet transplantation consistently yielded greater cell survival than cell injection. Immunohistochemistry revealed that cardiac cell sheets existed over the infarcted area as an intact layer. In contrast, the injected cells were scattered with relatively few cardiomyocytes in the infarcted areas. Four weeks after transplantation, cardiac function was also significantly improved in the cell sheet transplantation group compared with the cell injection. Twenty-four hours after cell grafting, significantly greater numbers of mature

  3. Influence of heavy ions on cell survival, cytogenetic damage and mitochondrial function of human endothelial cells

    NASA Astrophysics Data System (ADS)

    Ritter, Sylvia; Helm, Alexander; Lee, Ryonfa; Pollet, Dieter; Durante, Marco

    There is increasing evidence that there is an elevated risk of cardiovascular disease among atomic bomb survivors and radiotherapy patients, typically developing with a long latency. However, essentially no information is available on the potential cardiovascular risks associated with space radiation, in particular heavy ions. To address this issue, we have chosen human umbilical vein endothelial cells (HUVEC) as a model system. Cells at an early passage number were irradiated with 0.1 to 4 Gy of either 9.8 MeV/u C-ions (LET=170 keV/µm), 91 MeV/u C-ions (LET=29 keV/µm) or 250 kV X-rays. Cells were regularly subcultured up to 40 days (20 population doublings) post-irradiation. Immediately after exposure cell inactivation was deter-mined by the colony forming assay. Furthermore, at selected time-points cytogenetic damage (formation of micronuclei in binucleated cells) and the mitochondrial membrane potential ΨM (flow cytometric analysis following JC-1 staining) were assessed. Measurement of the directly induced radiation damage showed that 9.8 MeV/u and 91 MeV/u C-ions were more effective than X-rays (i.e. about 3 and 2 times, respectively) with respect to cell inactivation or the in-duction of cytogenetic damage. At the subsequent days in the irradiated cultures the number of cells with micronuclei declined to the control level (3-5Altogether our data indicate that under the applied radiation conditions the integrity of mitochondria which play a significant role in the regulation of cardiovascular cell function is not impaired. With respect to directly induced genetic damage C-ions are more effective than X-rays as observed in other cell systems. If the effectiveness of charged particles for the occurrence of late chromosomal damage in endothelial cells is higher than that of sparsely ionizing radiation needs further clarification. The data obtained up to now indicate that sophisticated cytogenetic techniques have to be applied in order to draw any firm

  4. Acovenoside A Induces Mitotic Catastrophe Followed by Apoptosis in Non-Small-Cell Lung Cancer Cells.

    PubMed

    El Gaafary, Menna; Ezzat, Shahira M; El Sayed, Abeer M; Sabry, Omar M; Hafner, Susanne; Lang, Sophia; Schmiech, Michael; Syrovets, Tatiana; Simmet, Thomas

    2017-12-22

    We investigated the cytotoxic potential of the cardenolide glycoside acovenoside A against non-small-cell lung cancer cells. Lung cancer is the leading cause of cancer-related mortality and the second most common cancer diagnosed. Epidemiological studies revealed a direct correlation between the regular administration of cardiac glycosides and a lower incidence of various cancers. Acovenoside A, isolated from the pericarps of Acokanthera oppositifolia, potently inhibited proliferation and induced cytotoxicity in A549 non-small-cell lung cancer cells with an IC 50 of 68 ± 3 nM after 48 h of exposure. Compared to the antineoplastic agent doxorubicin, acovenoside A was more potent in inhibiting the viability of A549 cancer cells. Moreover, acovenoside A exhibited selectivity against cancer cells, being significantly less toxic to lung fibroblasts and nontoxic for peripheral blood mononuclear cells. Analysis of the cell cycle profile in acovenoside A-treated A549 cells revealed mitotic arrest, due to accumulation of the G 2 /M regulators cyclin B 1 and CDK1, and cytokinesis failure. Furthermore, acovenoside A affected the mitochondrial membrane integrity and induced production of radical oxygen species, which resulted in induction of canonical apoptosis, manifested by caspase 3 activation and DNA fragmentation. Based on our results, acovenoside A warrants further exploration as a potential anticancer lead.

  5. Bacillus thuringiensis membrane-damaging toxins acting on mammalian cells.

    PubMed

    Celandroni, Francesco; Salvetti, Sara; Senesi, Sonia; Ghelardi, Emilia

    2014-12-01

    Bacillus thuringiensis is widely used as a biopesticide in forestry and agriculture, being able to produce potent species-specific insecticidal toxins and considered nonpathogenic to other animals. More recently, however, repeated observations are documenting the association of this microorganism with various infectious diseases in humans, such as food-poisoning-associated diarrheas, periodontitis, bacteremia, as well as ocular, burn, and wound infections. Similar to B. cereus, B. thuringiensis produces an array of virulence factors acting against mammalian cells, such as phosphatidylcholine- and phosphatidylinositol-specific phospholipase C (PC-PLC and PI-PLC), hemolysins, in particular hemolysin BL (HBL), and various enterotoxins. The contribution of some of these toxins to B. thuringiensis pathogenicity has been studied in animal models of infection, following intravitreous, intranasal, or intratracheal inoculation. These studies lead to the speculation that the activities of PC-PLC, PI-PLC, and HBL are responsible for most of the pathogenic properties of B. thuringiensis in nongastrointestinal infections in mammals. This review summarizes data regarding the biological activity, the genetic basis, and the structural features of these membrane-damaging toxins. © 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.

  6. SPARC (secreted protein acidic and rich in cysteine) knockdown protects mice from acute liver injury by reducing vascular endothelial cell damage

    PubMed Central

    Peixoto, E; Atorrasagasti, C; Aquino, JB; Militello, R; Bayo, J; Fiore, E; Piccioni, F; Salvatierra, E; Alaniz, L; García, MG; Bataller, R; Corrales, F; Gidekel, M; Podhajcer, O; Colombo, MI; Mazzolini, G

    2015-01-01

    Secreted protein, acidic and rich in cysteine (SPARC) is involved in many biological process including liver fibrogenesis, but its role in acute liver damage is unknown. To examine the role of SPARC in acute liver injury, we used SPARC knock-out (SPARC−/−) mice. Two models of acute liver damage were used: concanavalin A (Con A) and the agonistic anti-CD95 antibody Jo2. SPARC expression levels were analyzed in liver samples from patients with acute-on-chronic alcoholic hepatitis (AH). SPARC expression is increased on acute-on-chronic AH patients. Knockdown of SPARC decreased hepatic damage in the two models of liver injury. SPARC−/− mice showed a marked reduction in Con A-induced necroinflammation. Infiltration by CD4+ T cells, expression of tumor necrosis factor-α and interleukin-6 and apoptosis were attenuated in SPARC−/− mice. Sinusoidal endothelial cell monolayer was preserved and was less activated in Con A-treated SPARC−/− mice. SPARC knockdown reduced Con A-induced autophagy of cultured human microvascular endothelial cells (HMEC-1). Hepatic transcriptome analysis revealed several gene networks that may have a role in the attenuated liver damaged found in Con A-treated SPARC−/− mice. SPARC has a significant role in the development of Con A-induced severe liver injury. These results suggest that SPARC could represent a therapeutic target in acute liver injury. PMID:25410742

  7. [Research advances in the damage and regeneration of mammalian vestibular hair cells].

    PubMed

    Guo, J Y; Wang, G P; Gong, S S

    2017-01-07

    Vertigo is a common symptom in the clinic and impacts life quality of patients. It is closely related to the damage of vestibular hair cells. So far, there is no available approach which can facilitate abundant regeneration of mammalian vestibular hair cells, so as to recover the impaired vestibular function. Illuminating the mechanisms underlying vestibular hair cell damage and developing potential therapeutic strategies for vestibular hair cell regeneration are of great significance for the prevention and treatment of vertigo. In this study, we summarized research advances in the damage and regeneration of mammalian vestibular hair cells.

  8. Unrepaired DNA damage facilitates elimination of uniparental chromosomes in interspecific hybrid cells

    PubMed Central

    Wang, Zheng; Yin, Hao; Lv, Lei; Feng, Yingying; Chen, Shaopeng; Liang, Junting; Huang, Yun; Jiang, Xiaohua; Jiang, Hanwei; Bukhari, Ihtisham; Wu, Lijun; Cooke, Howard J; Shi, Qinghua

    2014-01-01

    Elimination of uniparental chromosomes occurs frequently in interspecific hybrid cells. For example, human chromosomes are always eliminated during clone formation when human cells are fused with mouse cells. However, the underlying mechanisms are still elusive. Here, we show that the elimination of human chromosomes in human–mouse hybrid cells is accompanied by continued cell division at the presence of DNA damage on human chromosomes. Deficiency in DNA damage repair on human chromosomes occurs after cell fusion. Furthermore, increasing the level of DNA damage on human chromosomes by irradiation accelerates human chromosome loss in hybrid cells. Our results indicate that the elimination of human chromosomes in human–mouse hybrid cells results from unrepaired DNA damage on human chromosomes. We therefore provide a novel mechanism underlying chromosome instability which may facilitate the understanding of carcinogenesis. PMID:24608870

  9. Orchestration of DNA Damage Checkpoint Dynamics across the Human Cell Cycle.

    PubMed

    Chao, Hui Xiao; Poovey, Cere E; Privette, Ashley A; Grant, Gavin D; Chao, Hui Yan; Cook, Jeanette G; Purvis, Jeremy E

    2017-11-22

    Although molecular mechanisms that prompt cell-cycle arrest in response to DNA damage have been elucidated, the systems-level properties of DNA damage checkpoints are not understood. Here, using time-lapse microscopy and simulations that model the cell cycle as a series of Poisson processes, we characterize DNA damage checkpoints in individual, asynchronously proliferating cells. We demonstrate that, within early G1 and G2, checkpoints are stringent: DNA damage triggers an abrupt, all-or-none cell-cycle arrest. The duration of this arrest correlates with the severity of DNA damage. After the cell passes commitment points within G1 and G2, checkpoint stringency is relaxed. By contrast, all of S phase is comparatively insensitive to DNA damage. This checkpoint is graded: instead of halting the cell cycle, increasing DNA damage leads to slower S phase progression. In sum, we show that a cell's response to DNA damage depends on its exact cell-cycle position and that checkpoints are phase-dependent, stringent or relaxed, and graded or all-or-none. Copyright © 2017 Elsevier Inc. All rights reserved.

  10. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells.

    PubMed

    Yedjou, Clement G; Tchounwou, Hervey M; Tchounwou, Paul B

    2015-12-22

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO₃)₂] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO₃)₂ for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO₃)₂-treated cells, indicative of membrane rupture by Pb(NO₃)₂ compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO₃)₂ exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO₃)₂ exposure caused cell cycle arrest at the G₀/G₁ checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO₃)₂ inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G₀/G₁ checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb

  11. DNA Damage, Cell Cycle Arrest, and Apoptosis Induction Caused by Lead in Human Leukemia Cells

    PubMed Central

    Yedjou, Clement G.; Tchounwou, Hervey M.; Tchounwou, Paul B.

    2015-01-01

    In recent years, the industrial use of lead has been significantly reduced from paints and ceramic products, caulking, and pipe solder. Despite this progress, lead exposure continues to be a significant public health concern. The main goal of this research was to determine the in vitro mechanisms of lead nitrate [Pb(NO3)2] to induce DNA damage, apoptosis, and cell cycle arrest in human leukemia (HL-60) cells. To reach our goal, HL-60 cells were treated with different concentrations of Pb(NO3)2 for 24 h. Live cells and necrotic death cells were measured by the propidium idiode (PI) assay using the cellometer vision. Cell apoptosis was measured by the flow cytometry and DNA laddering. Cell cycle analysis was evaluated by the flow cytometry. The result of the PI demonstrated a significant (p < 0.05) increase of necrotic cell death in Pb(NO3)2-treated cells, indicative of membrane rupture by Pb(NO3)2 compared to the control. Data generated from the comet assay indicated a concentration-dependent increase in DNA damage, showing a significant increase (p < 0.05) in comet tail-length and percentages of DNA cleavage. Data generated from the flow cytometry assessment indicated that Pb(NO3)2 exposure significantly (p < 0.05) increased the proportion of caspase-3 positive cells (apoptotic cells) compared to the control. The flow cytometry assessment also indicated Pb(NO3)2 exposure caused cell cycle arrest at the G0/G1 checkpoint. The result of DNA laddering assay showed presence of DNA smear in the agarose gel with little presence of DNA fragments in the treated cells compared to the control. In summary, Pb(NO3)2 inhibits HL-60 cells proliferation by not only inducing DNA damage and cell cycle arrest at the G0/G1 checkpoint but also triggering the apoptosis through caspase-3 activation and nucleosomal DNA fragmentation accompanied by secondary necrosis. We believe that our study provides a new insight into the mechanisms of Pb(NO3)2 exposure and its associated adverse health

  12. Raddeanin A induces human gastric cancer cells apoptosis and inhibits their invasion in vitro

    SciTech Connect

    Xue, Gang; Zou, Xi; Zhou, Jin-Yong

    2013-09-20

    Highlights: •Raddeanin A is a triterpenoid saponin in herb medicine Anemone raddeana Regel. •Raddeanin A can inhibit 3 kinds of gastric cancer cells’ proliferation and invasion. •Caspase-cascades’ activation indicates apoptosis induced by Raddeanin A. •MMPs, RECK, Rhoc and E-cad are involved in Raddeanin A-induced invasion inhibition. -- Abstract: Raddeanin A is one of the triterpenoid saponins in herbal medicine Anemone raddeana Regel which was reported to suppress the growth of liver and lung cancer cells. However, little was known about its effect on gastric cancer (GC) cells. This study aimed to investigate its inhibitory effect on three kinds of differentmore » differentiation stage GC cells (BGC-823, SGC-7901 and MKN-28) in vitro and the possible mechanisms. Proliferation assay and flow cytometry demonstrated Raddeanin A’s dose-dependent inhibitory effect and determined its induction of cells apoptosis, respectively. Transwell assay, wounding heal assay and cell matrix adhesion assay showed that Raddeanin A significantly inhibited the abilities of the invasion, migration and adhesion of the BGC-823 cells. Moreover, quantitative real time PCR and Western blot analysis found that Raddeanin A increased Bax expression while reduced Bcl-2, Bcl-xL and Survivin expressions and significantly activated caspase-3, caspase-8, caspase-9 and poly-ADP ribose polymerase (PARP). Besides, Raddeanin A could also up-regulate the expression of reversion inducing cysteine rich protein with Kazal motifs (RECK), E-cadherin (E-cad) and down-regulate the expression of matrix metalloproteinases-2 (MMP-2), MMP-9, MMP-14 and Rhoc. In conclusion, Raddeanin A inhibits proliferation of human GC cells, induces their apoptosis and inhibits the abilities of invasion, migration and adhesion, exhibiting potential to become antitumor drug.« less

  13. Interleukin-17A induces bicarbonate secretion in normal human bronchial epithelial cells

    PubMed Central

    Kreindler, James L.; Bertrand, Carol A.; Lee, Robert J.; Karasic, Thomas; Aujla, Shean; Pilewski, Joseph M.; Frizzell, Raymond A.; Kolls, Jay K.

    2009-01-01

    The innate immune functions of human airways include mucociliary clearance and antimicrobial peptide activity. Both functions may be affected by changes in epithelial ion transport. Interleukin-17A (IL-17A), which has a receptor at the basolateral membrane of airway epithelia, is a T cell cytokine that has been shown to increase mucus secretion and antimicrobial peptide production by human bronchial epithelial (HBE) cells. Furthermore, IL-17A levels are increased in sputum from patients during pulmonary exacerbations of cystic fibrosis. Therefore, we investigated the effects of IL-17A on basal, amiloride-sensitive, and forskolin-stimulated ion transport in mature, well-differentiated HBE cells. Exposure of HBE monolayers to IL-17A for 48 h induced a novel forskolin-stimulated bicarbonate secretion in addition to forskolin-stimulated chloride secretion and resulted in alkalinization of liquid on the mucosal surface of polarized cells. IL-17A-induced bicarbonate secretion was cystic fibrosis transmembrane conductance regulator (CFTR)-dependent, mucosal chloride-dependent, partially Na+-dependent, and sensitive to serosal, but not mucosal, stilbene inhibition. These data suggest that IL-17A modulates epithelial bicarbonate secretion and implicate a mechanism by which airway surface liquid pH changes may be abnormal in cystic fibrosis. PMID:19074559

  14. Genistein protects methylglyoxal-induced oxidative DNA damage and cell injury in human mononuclear cells.

    PubMed

    Wu, Hsin-Jung; Chan, Wen-Hsiung

    2007-04-01

    Methylglyoxal (MG) is a reactive dicarbonyl compound produced mainly from glycolytic intermediates in the cell and often found at high level in the blood from the diabetic patients. Glycation reactions of MG with amino acids can induce oxidative stress and free radical generation, leading to subsequent cytotoxicity and apoptosis. Recently, studies have demonstrated that high level MG may be the main cause of immune dysfunction in diabetic patients. Here, we examined the effects of genistein, an antioxidant isoflavone compound, on MG-induced effects in vitro and in human mononuclear cells. We first monitored DNA strand breakage to examine the effect of 12.5-100 microM genistein on the ROS generation and oxidative DNA damage induced in vitro by a 50 microM MG/lysine glycation reaction (3h). Our results revealed that genistein concentrations higher than 25 microM decreased the oxidative stress and DNA damage induced by 50 microM MG/lysine. In mononuclear cells, pretreatment with 8-16 microM genistein for 1h followed by co-incubation with genistein and 50 microM MG for an additional 36 h inhibited MG-induced reactive oxygen species (ROS) generation and apoptosis. Finally, animal model experiments showed that dietary genistein effectively blocks MG-induced apoptosis in mononuclear cells. These results collectively suggest that oxidative stress plays a role in MG-induced cell injury, and that genistein blocks these effects by virtue of its antioxidant properties, consequently preventing cell apoptosis.

  15. Retinal ganglion cell neuronal damage in diabetes and diabetic retinopathy.

    PubMed

    Ng, Dorothy Sk; Chiang, Peggy Pc; Tan, Gavin; Cheung, Cm Gemmy; Cheng, Ching-Yu; Cheung, Carol Y; Wong, Tien Y; Lamoureux, Ecosse L; Ikram, Mohammad K

    2016-05-01

    To examine the association of diabetes and diabetic retinopathy (DR) with retinal ganglion cell (RGC) loss. Observational case-control study. Type 2 diabetes cases and age-gender matched controls without diabetes. Spectral-domain optical coherence tomography (OCT) parameters of RGCs were calculated after automated segmentation of macular scans. DR severity was graded on fundus photographs using the modified Airlie House Classification system. Generalized estimating equation was used to compare OCT parameters between cases and controls, adjusted for covariates. Average ganglion cell-inner plexiform layer (GC-IPL) and average retinal nerve fibre layer (RNFL) thicknesses. We analyzed 227 cases and 227 controls. The mean age (years) of cases was 58.3 and controls was 58.1 (P = 0.13). Among cases, 101 had none, 25 had mild and 101 had moderate or severe DR. Compared with controls, GC-IPL and RNFL were thinner in all cases [mean difference (95% confidence interval [CI]): GC-IPL -4.49 µm (-2.92; -6.06), RNFL -0.93 µm (-0.09; -1.85)], including cases with no DR [mean difference (95% CI), GC-IPL -4.37 µm (-2.72; -6.02), RNFL -1.06 µm (-0.10; -2.02)]. Cases with any DR had thinner GC-IPL than controls [mean difference (95% CI): GC-IPL -4.81 µm (-2.12; -7.50)]. Among cases, subjects with moderate or severe DR had thinner GC-IPL than subjects with no DR [mean difference (95% CI): GC-IPL -2.07 µm (-0.08; -4.07)]. RGC loss is present in subjects with diabetes and no DR, and is progressive in moderate or severe DR. RGC neuronal damage in diabetes and DR can be clinically detected using OCT. © 2016 Royal Australian and New Zealand College of Ophthalmologists.

  16. [Protective effect of Astragalus polysaccharide on MRC-5 cells from oxidative damage induced by hydrogen peroxide].

    PubMed

    Chen, Juexiao; Wang, Guifen; Li, Li; Zhang, Pengxia

    2015-08-01

    To investigate the effects of Astragalus polysaccharide (APS) on the expressions of apurinic/apyrimidinic endonuclease/redox factor-1 (APE/Ref-1) and thioredoxin (TRX) in MRC-5 human embryo lung fibroblasts induced by hydrogen peroxide (H2O2) and explore the mechanism of APS protecting MRC-5 cells from oxidative damage. The MRC-5 cells were randomly divided into groups: the normal control group, groups induced by H2O2 of different concentrations, groups treated with 200, 400, 800 mg/L APS. The inhibitory rate of cell proliferation in H2O2-induced MRC-5 cells was measured by MTT assay to make sure the successful establishment of oxidative damage model. With the optimal concentration of H2O2 and different concentrations of APS on MRC-5 cells, we determined the optimal concentrate of APS to prevent oxidative damage in MRC-5 cells. The level of 8-hydroxy-2'-deoxyguanosine (8-OHdG) was detected by immunofluorescence staining. The apoptotic cells were identified by flow cytometry (FCM). The mRNA and protein levels of APE/Ref-1 and TRX were respectively detected by reverse transcription PCR and Western blotting. H2O2 induced oxidative damage in MRC-5 cells in a concentration-dependent manner. We chose the oxidative damage model induced by 800 μmol/L H2O2 for 24 hours to further study the protective mechanism of APS. Compared with H2O2 control groups, 200 mg/L APS significantly increased the protein level of APE/Ref-1 and TRX, decreased the content of 8-OHdG and the apoptosis of MRC-5 cells, and improved dramatically the cell viability. H2O2 can induce oxidative damage in MRC-5 cells. APS can promote APE/Ref-1 and TRX expressions in the damaged MRC-5 cells to relieve the oxidative damage and inhibit cell apoptosis.

  17. Zinc protects HepG2 cells against the oxidative damage and DNA damage induced by ochratoxin A

    SciTech Connect

    Zheng, Juanjuan; Zhang, Yu; Xu, Wentao, E-mail: xuwentaoboy@sina.com

    2013-04-15

    Oxidative stress and DNA damage are the most studied mechanisms by which ochratoxin A (OTA) induces its toxic effects, which include nephrotoxicity, hepatotoxicity, immunotoxicity and genotoxicity. Zinc, which is an essential trace element, is considered a potential antioxidant. The aim of this paper was to investigate whether zinc supplement could inhibit OTA-induced oxidative damage and DNA damage in HepG2 cells and the mechanism of inhibition. The results indicated that that exposure of OTA decreased the intracellular zinc concentration; zinc supplement significantly reduced the OTA-induced production of reactive oxygen species (ROS) and decrease in superoxide dismutase (SOD) activity but did notmore » affect the OTA-induced decrease in the mitochondrial membrane potential (Δψ{sub m}). Meanwhile, the addition of the zinc chelator N,N,N′,N′-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) strongly aggravated the OTA-induced oxidative damage. This study also demonstrated that zinc helped to maintain the integrity of DNA through the reduction of OTA-induced DNA strand breaks, 8-hydroxy-2′-deoxyguanosine (8-OHdG) formation and DNA hypomethylation. OTA increased the mRNA expression of metallothionein1-A (MT1A), metallothionein2-A (MT2A) and Cu/Zn superoxide dismutase (SOD1). Zinc supplement further enhanced the mRNA expression of MT1A and MT2A, but it had no effect on the mRNA expression of SOD1 and catalase (CAT). Zinc was for the first time proven to reduce the cytotoxicity of OTA through inhibiting the oxidative damage and DNA damage, and regulating the expression of zinc-associated genes. Thus, the addition of zinc can potentially be used to reduce the OTA toxicity of contaminated feeds. - Highlights: ► OTA decreased the intracellular zinc concentration. ► OTA induced the formation of 8-OHdG in HepG2 cells. ► It was testified for the first time that OTA induced DNA hypomethylation. ► Zinc protects against the oxidative damage and DNA damage

  18. Fanconi anemia cells with unrepaired DNA damage activate components of the checkpoint recovery process.

    PubMed

    Rodríguez, Alfredo; Torres, Leda; Juárez, Ulises; Sosa, David; Azpeitia, Eugenio; García-de Teresa, Benilde; Cortés, Edith; Ortíz, Rocío; Salazar, Ana M; Ostrosky-Wegman, Patricia; Mendoza, Luis; Frías, Sara

    2015-09-18

    The FA/BRCA pathway repairs DNA interstrand crosslinks. Mutations in this pathway cause Fanconi anemia (FA), a chromosome instability syndrome with bone marrow failure and cancer predisposition. Upon DNA damage, normal and FA cells inhibit the cell cycle progression, until the G2/M checkpoint is turned off by the checkpoint recovery, which becomes activated when the DNA damage has been repaired. Interestingly, highly damaged FA cells seem to override the G2/M checkpoint. In this study we explored with a Boolean network model and key experiments whether checkpoint recovery activation occurs in FA cells with extensive unrepaired DNA damage. We performed synchronous/asynchronous simulations of the FA/BRCA pathway Boolean network model. FA-A and normal lymphoblastoid cell lines were used to study checkpoint and checkpoint recovery activation after DNA damage induction. The experimental approach included flow cytometry cell cycle analysis, cell division tracking, chromosome aberration analysis and gene expression analysis through qRT-PCR and western blot. Computational simulations suggested that in FA mutants checkpoint recovery activity inhibits the checkpoint components despite unrepaired DNA damage, a behavior that we did not observed in wild-type simulations. This result implies that FA cells would eventually reenter the cell cycle after a DNA damage induced G2/M checkpoint arrest, but before the damage has been fixed. We observed that FA-A cells activate the G2/M checkpoint and arrest in G2 phase, but eventually reach mitosis and divide with unrepaired DNA damage, thus resolving the initial checkpoint arrest. Based on our model result we look for ectopic activity of checkpoint recovery components. We found that checkpoint recovery components, such as PLK1, are expressed to a similar extent as normal undamaged cells do, even though FA-A cells harbor highly damaged DNA. Our results show that FA cells, despite extensive DNA damage, do not loss the capacity to express

  19. From Attachment to Damage: Defined Genes of Candida albicans Mediate Adhesion, Invasion and Damage during Interaction with Oral Epithelial Cells

    PubMed Central

    Wächtler, Betty; Wilson, Duncan; Haedicke, Katja; Dalle, Frederic; Hube, Bernhard

    2011-01-01

    Candida albicans frequently causes superficial infections by invading and damaging epithelial cells, but may also cause systemic infections by penetrating through epithelial barriers. C. albicans is an unusual pathogen because it can invade epithelial cells via two distinct mechanisms: induced endocytosis, analogous to facultative intracellular enteropathogenic bacteria, and active penetration, similar to plant pathogenic fungi. Here we investigated the molecular basis of C. albicans epithelial interactions. By systematically assessing the contributions of defined fungal pathways and factors to different stages of epithelial interactions, we provide an expansive portrait of the processes and activities involved in epithelial infection. We strengthen the concept that hyphal formation is critical for epithelial invasion. Importantly, our data support a model whereby initial epithelial invasion per se does not elicit host damage, but that C. albicans relies on a combination of contact-sensing, directed hyphal extension, active penetration and the expression of novel pathogenicity factors for further inter-epithelial invasion, dissemination and ultimate damage of host cells. Finally, we explore the transcriptional landscape of C. albicans during the early stages of epithelial interaction, and, via genetic analysis, identify ICL1 and PGA34 as novel oral epithelial pathogenicity factors. PMID:21407800

  20. Semaphorin 3A Induces Odontoblastic Phenotype in Dental Pulp Stem Cells.

    PubMed

    Yoshida, S; Wada, N; Hasegawa, D; Miyaji, H; Mitarai, H; Tomokiyo, A; Hamano, S; Maeda, H

    2016-10-01

    In cases of pulp exposure due to deep dental caries or severe traumatic injuries, existing pulp-capping materials have a limited ability to reconstruct dentin-pulp complexes and can result in pulpectomy because of their low potentials to accelerate dental pulp cell activities, such as migration, proliferation, and differentiation. Therefore, the development of more effective therapeutic agents has been anticipated for direct pulp capping. Dental pulp tissues are enriched with dental pulp stem cells (DPSCs). Here, the authors investigated the effects of semaphorin 3A (Sema3A) on various functions of human DPSCs in vitro and reparative dentin formation in vivo in a rat dental pulp exposure model. Immunofluorescence staining revealed expression of Sema3A and its receptor Nrp1 (neuropilin 1) in rat dental pulp tissue and human DPSC clones. Sema3A induced cell migration, chemotaxis, proliferation, and odontoblastic differentiation of DPSC clones. In addition, Sema3A treatment of DPSC clones increased β-catenin nuclear accumulation, upregulated expression of the FARP2 gene (FERM, RhoGEF, and pleckstrin domain protein 2), and activated Rac1 in DPSC clones. Furthermore, in the rat dental pulp exposure model, Sema3A promoted reparative dentin formation with dentin tubules and a well-aligned odontoblast-like cell layer at the dental pulp exposure site and with novel reparative dentin almost completely covering pulp tissue at 4 wk after direct pulp capping. These findings suggest that Sema3A could play an important role in dentin regeneration via canonical Wnt/β-catenin signaling. Sema3A might be an alternative agent for direct pulp capping, which requires further study. © International & American Associations for Dental Research 2016.

  1. Vitamin E protects against the mitochondrial damage caused by cyclosporin A in LLC-PK1 cells

    SciTech Connect

    Arriba, G. de; Seccion de Nefrologia del Hospital Universitario de Guadalajara; Departamento de Medicina de la Universidad de Alcala de Henares

    2009-09-15

    Cyclosporin A (CsA) has nephrotoxic effects known to involve reactive oxygen species (ROS), since antioxidants prevent the kidney damage induced by this drug. Given that mitochondria are among the main sources of intracellular ROS, the aims of our study were to examine the mitochondrial effects of CsA in the porcine renal endothelial cell line LLC-PK1 and the influence of the antioxidant Vitamin E (Vit E). Following the treatment of LLC-PK1 cells with CsA, we assessed the mitochondrial synthesis of superoxide anion, permeability transition pore opening, mitochondrial membrane potential, cardiolipin peroxidation, cytochrome c release and cellular apoptosis, using flow cytometry andmore » confocal microscopy procedures. Similar experiments were done after Vit E preincubation of cells. CsA treatment increased superoxide anion in a dose-dependent way. CsA opened the permeability transition pores, caused Bax migration to mitochondria, and decreased mitochondrial membrane potential and cardiolipin content. Also CsA released cytochrome c into cytosol and provoked cellular apoptosis. Vit E pretreatment inhibited the effects that CsA induced on mitochondrial structure and function in LLC-PK1 cells and avoided apoptosis. CsA modifies mitochondrial LLC-PK1 cell physiology with loss of negative electrochemical gradient across the inner mitochondrial membrane and increased lipid peroxidation. These features are related to apoptosis and can explain the cellular damage that CsA induces. As Vit E inhibited these effects, our results suggest that they were mediated by an increase in ROS production by mitochondria.« less

  2. Cell to Cell Variability of Radiation-Induced Foci: Relation between Observed Damage and Energy Deposition.

    PubMed

    Gruel, Gaëtan; Villagrasa, Carmen; Voisin, Pascale; Clairand, Isabelle; Benderitter, Marc; Bottollier-Depois, Jean-François; Barquinero, Joan Francesc

    2016-01-01

    Most studies that aim to understand the interactions between different types of photon radiation and cellular DNA assume homogeneous cell irradiation, with all cells receiving the same amount of energy. The level of DNA damage is therefore generally determined by averaging it over the entire population of exposed cells. However, evaluating the molecular consequences of a stochastic phenomenon such as energy deposition of ionizing radiation by measuring only an average effect may not be sufficient for understanding some aspects of the cellular response to this radiation. The variance among the cells associated with this average effect may also be important for the behaviour of irradiated tissue. In this study, we accurately estimated the distribution of the number of radiation-induced γH2AX foci (RIF) per cell nucleus in a large population of endothelial cells exposed to 3 macroscopic doses of gamma rays from 60Co. The number of RIF varied significantly and reproducibly from cell to cell, with its relative standard deviation ranging from 36% to 18% depending on the macroscopic dose delivered. Interestingly, this relative cell-to-cell variability increased as the dose decreased, contrary to the mean RIF count per cell. This result shows that the dose effect, in terms of the number of DNA lesions indicated by RIF is not as simple as a purely proportional relation in which relative SD is constant with dose. To analyse the origins of this observed variability, we calculated the spread of the specific energy distribution for the different target volumes and subvolumes in which RIF can be generated. Variances, standard deviations and relative standard deviations all changed similarly from dose to dose for biological and calculated microdosimetric values. This similarity is an important argument that supports the hypothesis of the conservation of the association between the number of RIF per nucleus and the specific energy per DNA molecule. This comparison allowed us to

  3. Cell damage and dye reduction in the quantitative nitroblue tetrazolium (NBT) test.

    PubMed Central

    Segal, A W; Levi, A J

    1975-01-01

    Nitroblue tetrazolium (NBT) is toxic to neutrophils; an effect which is greatly enhanced by endotoxin and latex particles. Cell damage, measured by the release of the cytoplasmic marker enzyme lactate dehydrogenase (LDH), was closely related to dye reduction. This suggests that, in this test, dye reduction occurs largely as a result of contact between intracellular reducing compounds and NBT following damage of the outer cell membrane. The expression of dye reduction as a function of LDH release should enhance the sensitivity of the quantitative NBT test by correcting for the observed intersubject variation in cell damage. The relationship between cell damage and dye reduction is a measure of the reducing capacity of the cell. This was normal in immature, bone marrow neutrophils, but diminished in neutrophils of patients with chronic granulomatous disease. Images Fig. 3 PMID:1212802

  4. Ginsenoside Rg3 induces DNA damage in human osteosarcoma cells and reduces MNNG-induced DNA damage and apoptosis in normal human cells.

    PubMed

    Zhang, Yue-Hui; Li, Hai-Dong; Li, Bo; Jiang, Sheng-Dan; Jiang, Lei-Sheng

    2014-02-01

    Panax ginseng is a Chinese medicinal herb. Ginsenosides are the main bioactive components of P. ginseng, and ginsenoside Rg3 is the primary ginsenoside. Ginsenosides can potently kill various types of cancer cells. The present study was designed to evaluate the potential genotoxicity of ginsenoside Rg3 in human osteosarcoma cells and the protective effect of ginsenoside Rg3 with respect to N-methyl-N'-nitro-N-nitrosoguanidine (MNNG)-induced DNA damage and apoptosis in a normal human cell line (human fibroblasts). Four human osteosarcoma cell lines (MG-63, OS732, U-2OS and HOS cells) and a normal human cell line (human fibroblasts) were employed to investigate the cytotoxicity of ginsenosides Rg3 by MTT assay. Alkaline comet assay and γH2AX focus staining were used to detect the DNA damage in MG-63 and U-2OS cells. The extent of cell apoptosis was determined by flow cytometry and a DNA ladder assay. Our results demonstrated that the cytotoxicity of ginsenoside Rg3 was dose-dependent in the human osteosarcoma cell lines, and MG-63 and U-2OS cells were the most sensitive to ginsenoside Rg3. As expected, compared to the negative control, ginsenoside Rg3 significantly increased DNA damage in a concentration-dependent manner. In agreement with the comet assay data, the percentage of γH2AX-positive MG-63 and U-2OS cells indicated that ginsenoside Rg3 induced DNA double-strand breaks in a concentration-dependent manner. The results also suggest that ginsenoside Rg3 reduces the extent of MNNG-induced DNA damage and apoptosis in human fibroblasts.

  5. Clustered DNA damages induced in human hematopoietic cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, Betsy M.; Bennett, Paula V.; Cintron-Torres, Nela; Hada, Megumi; Trunk, John; Monteleone, Denise; Sutherland, John C.; Laval, Jacques; Stanislaus, Marisha; Gewirtz, Alan

    2002-01-01

    Ionizing radiation induces clusters of DNA damages--oxidized bases, abasic sites and strand breaks--on opposing strands within a few helical turns. Such damages have been postulated to be difficult to repair, as are double strand breaks (one type of cluster). We have shown that low doses of low and high linear energy transfer (LET) radiation induce such damage clusters in human cells. In human cells, DSB are about 30% of the total of complex damages, and the levels of DSBs and oxidized pyrimidine clusters are similar. The dose responses for cluster induction in cells can be described by a linear relationship, implying that even low doses of ionizing radiation can produce clustered damages. Studies are in progress to determine whether clusters can be produced by mechanisms other than ionizing radiation, as well as the levels of various cluster types formed by low and high LET radiation.

  6. Curcumin sensitizes lymphoma cells to DNA damage agents through regulating Rad51-dependent homologous recombination.

    PubMed

    Zhao, Qian; Guan, Jiawei; Qin, Yuanhua; Ren, Peng; Zhang, Zhiwei; Lv, Jian; Sun, Shijie; Zhang, Cuili; Mao, Weifeng

    2018-01-01

    Curcumin is a natural compound isolated from the rhizome of Curcuma longa. It possesses anti-tumor activity through arresting cell cycles and promoting cell apoptosis. However, the effect of curcumin on DNA damage is not well defined. In this study, we investigated the effect of curcumin on inducing DNA damage and on sensitizing lymphoma cells to anti-tumoral DNA damage drugs. Western blot showed curcumin induced γ-H2AX foci in CH12F3 lymphoma cells, which suggests curcumin induces DNA breaks. In addition, curcumin decreased the expression of Rad51, which suggests curcumin induces DNA damage through regulating Rad51-dependant homologous recombination. Rad51-dependant homologous recombination is a vital DNA repair pathway for cancer cells to resist anti-tumoral DNA damage drugs, therefore, we studied the effect of curcumin on the sensitizing lymphoma cells to various chemotherapeutic drugs. We found low level of curcumin (5μM) sensitized lymphoma cells to anti-tumoral DNA damage agents including cisplatin, methyl methanesulfonate, hydroxyurea and camptothecin. We also found curcumin sensitized CH12F3 lymphoma cells to DNA-PK and PARP inhibitors. Flow cytometry analysis showed curcumin promoted apoptosis and western blot analysis confirmed curcumin activated caspase3-dependent apoptosis. Taken together, these results demonstrate that curcumin induces DNA damage through regulating Rad51-dependant homologous recombination and triggers caspase3-dependent apoptosis, more importantly, curcumin sensitizes lymphoma cells to various DNA damage drugs. Consequently, curcumin would be a potent agent for sensitizing lymphoma cells to anti-tumoral chemotherapeutic agents. Copyright © 2017. Published by Elsevier Masson SAS.

  7. Regulated Cell Death of Lymphoma Cells after Graded Mitochondrial Damage is Differentially Affected by Drugs Targeting Cell Stress Responses.

    PubMed

    Lombardo, Tomás; Folgar, Martín Gil; Salaverry, Luciana; Rey-Roldán, Estela; Alvarez, Elida M; Carreras, María C; Kornblihtt, Laura; Blanco, Guillermo A

    2018-05-01

    Collapse of the mitochondrial membrane potential (MMP) is often considered the initiation of regulated cell death (RCD). Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) is an uncoupler of the electron transport chain (ETC) that facilitates the translocation of protons into the mitochondrial matrix leading to the collapse of the MMP. Several cell stress responses such as mitophagy, mitochondrial biogenesis and the ubiquitin proteasome system may differentially contribute to restrain the initiation of RCD depending on the extent of mitochondrial damage. We induced graded mitochondrial damage after collapse of MMP with the mitochondrial uncoupler CCCP in Burkitt's lymphoma cells, and we evaluated the effect of several drugs targeting cell stress responses over RCD at 72 hr, using a multiparametric flow cytometry approach. CCCP caused collapse of MMP after 30 min., massive mitochondrial fission, oxidative stress and increased mitophagy within the 5-15 μM low-dose range (LDR) of CCCP. Within the 20-50 μM high-dose range (HDR), CCCP caused lysosomal destabilization and rupture, thus precluding mitophagy and autophagy. Cell death after 72 hr was below 20%, with increased mitochondrial mass (MM). The inhibitors of mitophagy 3-(2,4-dichloro-5-methoxyphenyl)-2,3-dihydro-2-thioxo-4(1H)-quinazolinone (Mdivi-1) and vincristine (VCR) increased cell death from CCCP within the LDR, while valproic acid (an inducer of mitochondrial biogenesis) also increased MM and cell death within the LDR. The proteasome inhibitor, MG132, increased cell death only in the HDR. Doxycycline, an antibiotic that disrupts mitochondrial biogenesis, had no effect on cell survival, while iodoacetamide, an inhibitor of glycolysis, increased cell death at the HDR. We conclude that mitophagy influenced RCD of lymphoma cells after MMP collapse by CCCP only within the LDR, while proteasome activity and glycolysis contributed to survival in the HDR under extensive mitochondria and lysosome damage. © 2017

  8. The real deal: using cytochalasin B in sonodynamic therapy to preferentially damage leukemia cells.

    PubMed

    Trendowski, Matthew; Yu, Guowu; Wong, Victoria; Acquafondata, Christopher; Christen, Timothy; Fondy, Thomas P

    2014-05-01

    Sonodynamic therapy (SDT) is a form of ultrasound therapy in which chemotherapeutic agents known as sonosensitizers are administered to increase the efficacy of ultrasound's preferential damage to neoplastic cells. Perhaps one of the most intriguing capabilities of ultrasound is its ability to preferentially lyse cells based on size. Cytochalasin B is a cytokinesis inhibitor that preferentially enlarges and multinucleates malignant cells, making them much more sensitive to ultrasonic irradiation. The present study investigated the extent of preferential damage inflicted by cytochalasin B on U937 leukemia/human blood cell populations. Cell mixtures were treated with cytochalasin B and then sonicated under a relatively low intensity (3W/cm(2)). Cytochalasin B preferentially damages U937 cells both before and after sonication. This agent also reduces rapid proliferation as the clonogenicity of U937 cells was considerably reduced following treatment. Cytochalasin B may have profound therapeutic applications when combined with SDT.

  9. Automated High-Throughput Damage Scoring of Zebrafish Lateral Line Hair Cells After Ototoxin Exposure.

    PubMed

    Philip, Rohit C; Rodriguez, Jeffrey J; Niihori, Maki; Francis, Ross H; Mudery, Jordan A; Caskey, Justin S; Krupinski, Elizabeth; Jacob, Abraham

    2018-04-01

    Zebrafish have emerged as a powerful biological system for drug development against hearing loss. Zebrafish hair cells, contained within neuromasts along the lateral line, can be damaged with exposure to ototoxins, and therefore, pre-exposure to potentially otoprotective compounds can be a means of identifying promising new drug candidates. Unfortunately, anatomical assays of hair cell damage are typically low-throughput and labor intensive, requiring trained experts to manually score hair cell damage in fluorescence or confocal images. To enhance throughput and consistency, our group has developed an automated damage-scoring algorithm based on machine-learning techniques that produce accurate damage scores, eliminate potential operator bias, provide more fidelity in determining damage scores that are between two levels, and deliver consistent results in a fraction of the time required for manual analysis. The system has been validated against trained experts using linear regression, hypothesis testing, and the Pearson's correlation coefficient. Furthermore, performance has been quantified by measuring mean absolute error for each image and the time taken to automatically compute damage scores. Coupling automated analysis of zebrafish hair cell damage to behavioral assays for ototoxicity produces a novel drug discovery platform for rapid translation of candidate drugs into preclinical mammalian models of hearing loss.

  10. Docosahexaenoic Acid Induces Oxidative DNA Damage and Apoptosis, and Enhances the Chemosensitivity of Cancer Cells.

    PubMed

    Song, Eun Ah; Kim, Hyeyoung

    2016-08-03

    The human diet contains low amounts of ω-3 polyunsaturated fatty acids (PUFAs) and high amounts of ω-6 PUFAs, which has been reported to contribute to the incidence of cancer. Epidemiological studies have shown that a high consumption of fish oil or ω-3 PUFAs reduced the risk of colon, pancreatic, and endometrial cancers. The ω-3 PUFA, docosahexaenoic acid (DHA), shows anticancer activity by inducing apoptosis of some human cancer cells without toxicity against normal cells. DHA induces oxidative stress and oxidative DNA adduct formation by depleting intracellular glutathione (GSH) and decreasing the mitochondrial function of cancer cells. Oxidative DNA damage and DNA strand breaks activate DNA damage responses to repair the damaged DNA. However, excessive DNA damage beyond the capacity of the DNA repair processes may initiate apoptotic signaling pathways and cell cycle arrest in cancer cells. DHA shows a variable inhibitory effect on cancer cell growth depending on the cells' molecular properties and degree of malignancy. It has been shown to affect DNA repair processes including DNA-dependent protein kinases and mismatch repair in cancer cells. Moreover, DHA enhanced the efficacy of anticancer drugs by increasing drug uptake and suppressing survival pathways in cancer cells. In this review, DHA-induced oxidative DNA damage, apoptotic signaling, and enhancement of chemosensitivity in cancer cells will be discussed based on recent studies.

  11. Modelling the damage potential of fluid flows for animal cells undergoing cultivation in bioreactors.

    NASA Astrophysics Data System (ADS)

    Stanford Keen, Giles

    1996-11-01

    Mechanical disruption and injury sustained by animal cells undergoing cultivation in bioreactors is an important problem in biotechnology. Damage to cells is thought to be caused primarily by bubbles bursting at the free surface of the culture medium. Here we present computational studies applying a mathematical model for the cell damage rates experienced by cells in laminar flow. Two fluid dynamical systems are considered - namely a converging channel and a single bursting bubble. The flows are calculated using a fourth-order finite difference technique on a stretched grid, and a boundary integral method respectively. It is possible to obtain an estimate for the number of cells in a particular population which are likely to be disrupted by the forces they experience in the flow. This is done by calculating the maximum rate of strain experienced by fluid particles, and combining this with experimental data on the strength and size of cells, obtained by micromanipulation techniques. The resulting information is then used together with the cell damage model to produce a cell damage prediction. The computational results are compared with experimental measurements of cell death, to validate the model for cell damage.

  12. The same, only different - DNA damage checkpoints and their reversal throughout the cell cycle.

    PubMed

    Shaltiel, Indra A; Krenning, Lenno; Bruinsma, Wytse; Medema, René H

    2015-02-15

    Cell cycle checkpoints activated by DNA double-strand breaks (DSBs) are essential for the maintenance of the genomic integrity of proliferating cells. Following DNA damage, cells must detect the break and either transiently block cell cycle progression, to allow time for repair, or exit the cell cycle. Reversal of a DNA-damage-induced checkpoint not only requires the repair of these lesions, but a cell must also prevent permanent exit from the cell cycle and actively terminate checkpoint signalling to allow cell cycle progression to resume. It is becoming increasingly clear that despite the shared mechanisms of DNA damage detection throughout the cell cycle, the checkpoint and its reversal are precisely tuned to each cell cycle phase. Furthermore, recent findings challenge the dogmatic view that complete repair is a precondition for cell cycle resumption. In this Commentary, we highlight cell-cycle-dependent differences in checkpoint signalling and recovery after a DNA DSB, and summarise the molecular mechanisms that underlie the reversal of DNA damage checkpoints, before discussing when and how cell fate decisions after a DSB are made. © 2015. Published by The Company of Biologists Ltd.

  13. DNA damage and mitochondria dysfunction in cell apoptosis induced by nonthermal air plasma

    NASA Astrophysics Data System (ADS)

    Kim, G. J.; Kim, W.; Kim, K. T.; Lee, J. K.

    2010-01-01

    Nonthermal plasma is known to induce animal cell death but the mechanism is not yet clear. Here, cellular and biochemical regulation of cell apoptosis is demonstrated for plasma treated cells. Surface type nonthermal air plasma triggered apoptosis of B16F10 mouse melanoma cancer cells causing DNA damage and mitochondria dysfunction. Plasma treatment activated caspase-3, apoptosis executioner. The plasma treated cells also accumulated gamma-H2A.X, marker for DNA double strand breaks, and p53 tumor suppressor gene as a response to DNA damage. Interestingly, cytochrome C was released from mitochondria and its membrane potential was changed significantly.

  14. Dynamics of the DNA damage response: insights from live-cell imaging

    PubMed Central

    Karanam, Ketki; Loewer, Alexander

    2013-01-01

    All organisms have to safeguard the integrity of their genome to prevent malfunctioning and oncogenic transformation. Sophisticated DNA damage response mechanisms have evolved to detect and repair genomic lesions. With the emergence of live-cell microscopy of individual cells, we now begin to appreciate the complex spatiotemporal kinetics of the DNA damage response and can address the causes and consequences of the heterogeneity in the responses of genetically identical cells. Here, we highlight key discoveries where live-cell imaging has provided unprecedented insights into how cells respond to DNA double-strand breaks and discuss the main challenges and promises in using this technique. PMID:23292635

  15. [Neuroprotective effects of oligodendrocyte precursor cells on white matter damage in preterm infants].

    PubMed

    Yue, Yan; Zhang, Li; Qu, Yi; Mu, De-Zhi

    2018-04-01

    White matter damage, characterized by demyelination due to the damage of oligodendrocyte precursor cells (OPCs), is the most common type of brain damage in preterm infants. Survivors are often subject to long-term neurodevelopmental sequelae because of the lack of effective treatment. In recent years, it has been found that cell transplantation has the potential for the treatment of white matter damage. OPCs are frequently used cells in cell transplantation therapy. With abilities of migration and myelinization, OPCs are the best seed cells for the treatment of white matter damage. Several studies have found that OPCs may not only replace impaired cells to reconstruct the structure and function of white matter, but also inhibit neuronal apoptosis, promote the proliferation of endogenous neural stem cells, and enhance the repairment of the blood-brain barrier. However, the clinical application of OPC transplantation therapy faces many challenges, such as the effectiveness, risk of tumorigenesis and immune rejection. With reference to these studies, this article reviewed the development of myelination, the obtainment of OPCs, the therapeutic mechanism as well as application research, and analyzed the current challenges of OPC transplantation, in order to provide a new direction for clinical treatment of white matter damage in preterm infants.

  16. Vorinostat Induces Reactive Oxygen Species and DNA Damage in Acute Myeloid Leukemia Cells

    PubMed Central

    Pettersson, Filippa; Retrouvey, Hélène; Skoulikas, Sophia; Miller, Wilson H.

    2011-01-01

    Histone deacetylase inhibitors (HDACi) are promising anti-cancer agents, however, their mechanisms of action remain unclear. In acute myeloid leukemia (AML) cells, HDACi have been reported to arrest growth and induce apoptosis. In this study, we elucidate details of the DNA damage induced by the HDACi vorinostat in AML cells. At clinically relevant concentrations, vorinostat induces double-strand breaks and oxidative DNA damage in AML cell lines. Additionally, AML patient blasts treated with vorinostat display increased DNA damage, followed by an increase in caspase-3/7 activity and a reduction in cell viability. Vorinostat-induced DNA damage is followed by a G2-M arrest and eventually apoptosis. We found that pre-treatment with the antioxidant N-acetyl cysteine (NAC) reduces vorinostat-induced DNA double strand breaks, G2-M arrest and apoptosis. These data implicate DNA damage as an important mechanism in vorinostat-induced growth arrest and apoptosis in both AML cell lines and patient-derived blasts. This supports the continued study and development of vorinostat in AMLs that may be sensitive to DNA-damaging agents and as a combination therapy with ionizing radiation and/or other DNA damaging agents. PMID:21695163

  17. Small cell lung cancer: Time to revisit DNA-damaging chemotherapy.

    PubMed

    Thomas, Anish; Pommier, Yves

    2016-07-06

    Rational use of DNA-damaging chemotherapy, with new combinations to heighten DNA replication stress, could improve outcomes in small cell lung cancer. Copyright © 2016, American Association for the Advancement of Science.

  18. Endothelial–cell FAK targeting sensitizes tumours to DNA–damaging therapy

    PubMed Central

    Tavora, Bernardo; Reynolds, Louise E.; Batista, Silvia; Demircioglu, Fevzi; Fernandez, Isabelle; Lechertier, Tanguy; Lees, Delphine M.; Wong, Ping–Pui; Alexopoulou, Annika; Elia, George; Clear, Andrew; Ledoux, Adeline; Hunter, Jill; Perkins, Neil; Gribben, John G.; Hodivala–Dilke, Kairbaan M.

    2015-01-01

    Chemoresistance is a serious limitation of cancer treatment1. Until recently, almost all the work done to study this limitation has been restricted to tumour cells2. Here we identify a novel molecular mechanism by which endothelial cells regulate chemosensitivity. We establish that specific targeting of focal adhesion kinase (FAK; also known as PTK2) in endothelial cells is sufficient to induce tumour-cell sensitization to DNA-damaging therapies and thus inhibit tumour growth in mice. The clinical relevance of this work is supported by our observations that low blood vessel FAK expression is associated with complete remission in human lymphoma. Our study shows that deletion of FAK in endothelial cells has no apparent effect on blood vessel function per se, but induces increased apoptosis and decreased proliferation within perivascular tumour-cell compartments of doxorubicin- and radiotherapy-treated mice. Mechanistically, we demonstrate that endothelial-cell FAK is required for DNA-damage-induced NF-κB activation in vivo and in vitro, and the production of cytokines from endothelial cells. Moreover, loss of endothelial-cell FAK reduces DNA-damage-induced cytokine production, thus enhancing chemosensitization of tumour cells to DNA-damaging therapies in vitro and in vivo. Overall, our data identify endothelial-cell FAK as a regulator of tumour chemosensitivity. Furthermore, we anticipate that this proof-of-principle data will be a starting point for the development of new possible strategies to regulate chemosensitization by targeting endothelial-cell FAK specifically. PMID:25079333

  19. Nuclear translocation of phosphorylated STAT3 regulates VEGF-A-induced lymphatic endothelial cell migration and tube formation

    SciTech Connect

    Okazaki, Hideki; Tokumaru, Sho; Hanakawa, Yasushi

    2011-09-02

    Highlights: {yields} VEGF-A enhanced lymphatic endothelial cell migration and increased tube formation. {yields} VEGF-A treated lymphatic endothelial cell showed activation of STAT3. {yields} Dominant-negative STAT3 inhibited VEGF-A-induced lymphatic endothelial cell migration and tube formation. -- Abstract: Vascular endothelial growth factor (VEGF) is an endothelial cell-specific growth factor that regulates endothelial functions, and signal transducers and activators of transcription (STATs) are known to be important during VEGF receptor signaling. The aim of this study was to determine whether STAT3 regulates VEGF-induced lymphatic endothelial cell (LEC) migration and tube formation. VEGF-A (33 ng/ml) enhanced LEC migration by 2-fold and increased tube lengthmore » by 25% compared with the control, as analyzed using a Boyden chamber and Matrigel assay, respectively. Western blot analysis and immunostaining revealed that VEGF-A induced the nuclear translocation of phosphorylated STAT3 in LECs, and this translocation was blocked by the transfection of LECs with an adenovirus vector expressing a dominant-negative mutant of STAT3 (Ax-STAT3F). Transfection with Ax-STAT3F also almost completely inhibited VEGF-A-induced LEC migration and tube formation. These results indicate that STAT3 is essential for VEGF-A-induced LEC migration and tube formation and that STAT3 regulates LEC functions.« less

  20. Evaluating chromosome damage to estimate dose to tracheal epithelial cells

    SciTech Connect

    Brooks, A.L.; Rithidech, K.; Kitchin, R.M.

    1992-12-31

    Chromosome aberration frequency has been widely used to estimate dose in radiation accidents when no physical dosimetry was available. Basal and secretory cells are thought to represent the major cells at risk in the upper respiratory tract for induction of cancer. Using an in vivo/in vitro approach, we have evaluated chromosome aberrations to determine radiation sensitivity of basal and secretory cells and to estimate the dose to tracheal epithelial cells following radon inhalation. Tracheal epithelial cells were isolated, sorted by flow cytometry into populations of basal and secretory cells, grown in serum-free medium, and exposed to graded doses of xmore » rays. Chromosome aberration frequency was determined in both types of cells. A 2-Gy dose of x rays induced 0.52 {+-} 0.07 or 0.55 {+-} 0.07 aberrations/cells in the basal and secretory cells, respectively. These data suggest that the radiation sensitivity is similar for the two cell populations and, because these are the cells that divide, the measured aberration frequencies can provide an estimate of radiation dose. After evaluating the response to x rays it was essential to determine the dose-response relationship for tracheal epithelial cells exposed to alpha particles. Tracheal cells were isolated from the trachea, allowed to attach to a 3-{mu}m Mylar film, exposed to a {sup 238}Pu source, and grown in culture. The frequency of chromosome aberrations was then determined. One gray of alpha exposure produced 0.55 {+-} 0.06 aberrations/cell as well as 0.11 {+-} 0.04 dicentrics plus rings/cell. This provides a benchmark against which radon-induced aberrations in the trachea cells exposed to radon can be compared. In other experiments, two groups of F-344/N rats inhaled radon and its progeny at concentrations and times to produce total exposures of either 900 or 1000 working level months (WLM).« less

  1. Progressive glomerular and tubular damage in sickle cell trait and sickle cell anemia mouse models.

    PubMed

    Saraf, Santosh L; Sysol, Justin R; Susma, Alexandru; Setty, Suman; Zhang, Xu; Gudehithlu, Krishnamurthy P; Arruda, Jose A L; Singh, Ashok K; Machado, Roberto F; Gordeuk, Victor R

    2018-02-02

    Homozygosity for the hemoglobin (Hb) S mutation (HbSS, sickle cell anemia) results in hemoglobin polymerization under hypoxic conditions leading to vaso-occlusion and hemolysis. Sickle cell anemia affects 1:500 African Americans and is a strong risk factor for kidney disease, although the mechanisms are not well understood. Heterozygous inheritance (HbAS; sickle cell trait) affects 1:10 African Americans and is associated with an increased risk for kidney disease in some reports. Using transgenic sickle mice, we investigated the histopathologic, ultrastructural, and gene expression differences with the HbS mutation. Consistent with progressive glomerular damage, we observed progressively greater urine protein concentrations (P = 0.03), glomerular hypertrophy (P = 0.002), and glomerular cellularity (P = 0.01) in HbAA, HbAS, and HbSS mice, respectively. Ultrastructural studies demonstrated progressive podocyte foot process effacement, glomerular basement membrane thickening with reduplication, and tubular villous atrophy with the HbS mutation. Gene expression studies highlighted the differential expression of several genes involved in prostaglandin metabolism (AKR1C18), heme and iron metabolism (HbA-A2, HMOX1, SCL25A37), electrolyte balance (SLC4A1, AQP6), immunity (RSAD2, C3, UBE2O), fatty acid metabolism (FASN), hypoxia hall-mark genes (GCK, SDC3, VEGFA, ETS1, CP, BCL2), as well as genes implicated in other forms of kidney disease (PODXL, ELMO1, FRMD3, MYH9, APOA1). Pathway analysis highlighted increased gene enrichment in focal adhesion, extracellular matrix-receptor interaction, and axon guidance pathways. In summary, using transgenic sickle mice, we observed that inheritance of the HbS mutation is associated with glomerular and tubular damage and identified several candidate genes and pathways for future investigation in sickle cell trait and sickle cell anemia-related kidney disease. Copyright © 2018 Elsevier Inc. All rights reserved.

  2. Spatial Pattern of Cell Damage in Tissue from Heavy Ions

    NASA Technical Reports Server (NTRS)

    Ponomarev, Artem L.; Huff, Janice L.; Cucinotta, Francis A.

    2007-01-01

    A new Monte Carlo algorithm was developed that can model passage of heavy ions in a tissue, and their action on the cellular matrix for 2- or 3-dimensional cases. The build-up of secondaries such as projectile fragments, target fragments, other light fragments, and delta-rays was simulated. Cells were modeled as a cell culture monolayer in one example, where the data were taken directly from microscopy (2-d cell matrix). A simple model of tissue was given as abstract spheres with close approximation to real cell geometries (3-d cell matrix), as well as a realistic model of tissue was proposed based on microscopy images. Image segmentation was used to identify cells in an irradiated cell culture monolayer, or slices of tissue. The cells were then inserted into the model box pixel by pixel. In the case of cell monolayers (2-d), the image size may exceed the modeled box size. Such image was is moved with respect to the box in order to sample as many cells as possible. In the case of the simple tissue (3-d), the tissue box is modeled with periodic boundary conditions, which extrapolate the technique to macroscopic volumes of tissue. For real tissue, specific spatial patterns for cell apoptosis and necrosis are expected. The cell patterns were modeled based on action cross sections for apoptosis and necrosis estimated based on BNL data, and other experimental data.

  3. Comparative analysis of different laser systems to study cellular responses to DNA damage in mammalian cells

    PubMed Central

    Kong, Xiangduo; Mohanty, Samarendra K.; Stephens, Jared; Heale, Jason T.; Gomez-Godinez, Veronica; Shi, Linda Z.; Kim, Jong-Soo; Yokomori, Kyoko; Berns, Michael W.

    2009-01-01

    Proper recognition and repair of DNA damage is critical for the cell to protect its genomic integrity. Laser microirradiation ranging in wavelength from ultraviolet A (UVA) to near-infrared (NIR) can be used to induce damage in a defined region in the cell nucleus, representing an innovative technology to effectively analyze the in vivo DNA double-strand break (DSB) damage recognition process in mammalian cells. However, the damage-inducing characteristics of the different laser systems have not been fully investigated. Here we compare the nanosecond nitrogen 337 nm UVA laser with and without bromodeoxyuridine (BrdU), the nanosecond and picosecond 532 nm green second-harmonic Nd:YAG, and the femtosecond NIR 800 nm Ti:sapphire laser with regard to the type(s) of damage and corresponding cellular responses. Crosslinking damage (without significant nucleotide excision repair factor recruitment) and single-strand breaks (with corresponding repair factor recruitment) were common among all three wavelengths. Interestingly, UVA without BrdU uniquely produced base damage and aberrant DSB responses. Furthermore, the total energy required for the threshold H2AX phosphorylation induction was found to vary between the individual laser systems. The results indicate the involvement of different damage mechanisms dictated by wavelength and pulse duration. The advantages and disadvantages of each system are discussed. PMID:19357094

  4. Thermal analysis of induced damage to the healthy cell during RFA of breast tumor.

    PubMed

    Singh, Sundeep; Bhowmik, Arka; Repaka, Ramjee

    2016-05-01

    Effective pre-clinical computational modeling strategies have been demonstrated in this article to enable risk free clinical application of radiofrequency ablation (RFA) of breast tumor. The present study (a) determines various optimal regulating parameters required for RFA of tumor and (b) introduces an essential clinical monitoring scheme to minimize the extent of damage to the healthy cell during RFA of tumor. The therapeutic capabilities offered by RFA of breast tumor, viz., the rise in local temperature and induced thermal damage have been predicted by integrating the bioheat transfer model, the electric field distribution model and the thermal damage model. The mathematical model has been validated with the experimental results available in the literature. The results revealed that, the effective damage of tumor volume sparing healthy tissue essentially depends on the voltage, the exposure time, the local heat distribution, the tumor stage and the electrode geometric configuration. It has been confirmed that, the assessment of damage front can accurately determine the extent of damage as compared to the thermal front. The study further evaluates the damaged healthy and tumor volumes due to RFA of different stages of breast cancer. The assessment of cell survival and damage fractions discloses the propensity of reappearance/healing of tumor cells after treatment. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Depletion layer recombination effects on the radiation damage hardness of gallium arsenide cells

    NASA Technical Reports Server (NTRS)

    Garlick, G. F. J.

    1985-01-01

    The significant effect of junction depletion layer recombination on the efficiency of windowed GaAs cells was demonstrated. The effect becomes more pronounced as radiation damage occurs. The depletion is considered for 1 MeV electron fluences up to 10 to the 16th power e/sq m. The cell modeling separates damage in emitter and base or buffer layers using different damage coefficients is reported. The lower coefficient for the emitter predicts less loss of performance at fluences greater than 10 to the 15th power e/sq cm. A method for obtaining information on junction recombination effects as damage proceeds is described; this enables a more complete diagnosis of damage to be made.

  6. Radiation damage in high voltage silicon solar cells

    NASA Technical Reports Server (NTRS)

    Weinberg, I.; Brandhorst, H., Jr.; Swartz, C. K.; Weizer, V. G.

    1980-01-01

    Three high open-circuit voltage cell designs based on 0.1 ohm-cm p-type silicon were irradiated with 1 MeV electrons and their performance determined to fluences as high as 10 to the 15th power/sq cm. Of the three cell designs, radiation induced degradation was greatest in the high-low emitter (HLE cell). The diffused and ion implanted cells degraded approximately equally but less than the HLE cell. Degradation was greatest in an HLE cell exposed to X-rays before electron irradiation. The cell regions controlling both short-circuit current and open-circuit voltage degradation were defined in all three cell types. An increase in front surface recombination velocity accompanied time dependent degradation of an HLE cell after X-irradiation. It was speculated that this was indirectly due to a decrease in positive charge at the silicon-oxide interface. Modifications aimed at reducing radiation induced degradation are proposed for all three cell types.

  7. Correlation of electron and proton irradiation-induced damage in InP solar cells

    NASA Technical Reports Server (NTRS)

    Walters, Robert J.; Summers, Geoffrey P.; Messenger, Scott R.; Burke, Edward A.

    1996-01-01

    The measured degradation of epitaxial shallow homojunction n(+)/p InP solar cells under 1 MeV electron irradiation is correlated with that measured under 3 MeV proton irradiation based on 'displacement damage dose'. The measured data is analyzed as a function of displacement damage dose from which an electron to proton dose equivalency ratio is determined which enables the electron and proton degradation data to be described by a single degradation curve. It is discussed how this single curve can be used to predict the cell degradation under irradiation by any particle energy. The degradation curve is used to compare the radiation response of InP and GaAs/Ge cells on an absolute damage energy scale. The comparison shows InP to be inherently more resistant to displacement damage deposition than the GaAs/Ge.

  8. Plant Nuclei Move to Escape Ultraviolet-Induced DNA Damage and Cell Death1[OPEN

    PubMed Central

    Hidema, Jun; Tamura, Kentaro

    2016-01-01

    A striking feature of plant nuclei is their light-dependent movement. In Arabidopsis (Arabidopsis thaliana) leaf mesophyll cells, the nuclei move to the side walls of cells within 1 to 3 h after blue-light reception, although the reason is unknown. Here, we show that the nuclear movement is a rapid and effective strategy to avoid ultraviolet B (UVB)-induced damages. Mesophyll nuclei were positioned on the cell bottom in the dark, but sudden exposure of these cells to UVB caused severe DNA damage and cell death. The damage was remarkably reduced in both blue-light-treated leaves and mutant leaves defective in the actin cytoskeleton. Intriguingly, in plants grown under high-light conditions, the mesophyll nuclei remained on the side walls even in the dark. These results suggest that plants have two strategies for reducing UVB exposure: rapid nuclear movement against acute exposure and nuclear anchoring against chronic exposure. PMID:26681797

  9. Human cells enter mitosis with damaged DNA after treatment with pharmacological concentrations of genotoxic agents

    PubMed Central

    Kubara, Philip M.; Kernéis-Golsteyn, Sophie; Studény, Aurélie; Lanser, Brittany B.; Meijer, Laurent; Golsteyn, Roy M.

    2012-01-01

    In the present paper, we report that mitosis is a key step in the cellular response to genotoxic agents in human cells. Cells with damaged DNA recruit γH2AX (phosphorylated histone H2AX), phosphorylate Chk1 (checkpoint kinase 1) and arrest in the G2-phase of the cell cycle. Strikingly, nearly all cells escape the DNA damage checkpoint and become rounded, by a mechanism that correlates with Chk1 dephosphorylation. The rounded cells are alive and in mitosis as measured by low phospho-Tyr15 Cdk1 (cyclin-dependent kinase 1), high Cdk activity, active Plk1 (Polo-like kinase 1) and high phospho-histone H3 signals. This phenomenon is independent of the type of DNA damage, but is dependent on pharmacologically relevant doses of genotoxicity. Entry into mitosis is likely to be caused by checkpoint adaptation, and the HT-29 cell-based model provides a powerful experimental system in which to explore its molecular basis. We propose that mitosis with damaged DNA is a biologically significant event because it may cause genomic rearrangement in cells that survive genotoxic damage. PMID:22686412

  10. Mefloquine damage vestibular hair cells in organotypic cultures.

    PubMed

    Yu, Dongzhen; Ding, Dalian; Jiang, Haiyan; Stolzberg, Daniel; Salvi, Richard

    2011-07-01

    Mefloquine is an effective and widely used anti-malarial drug; however, some clinical reports suggest that it can cause dizziness, balance, and vestibular disturbances. To determine if mefloquine might be toxic to the vestibular system, we applied mefloquine to organotypic cultures of the macula of the utricle from postnatal day 3 rats. The macula of the utricle was micro-dissected out as a flat surface preparation and cultured with 10, 50, 100, or 200 μM mefloquine for 24 h. Specimens were stained with TRITC-conjugated phalloidin to label the actin in hair cell stereocilia and TO-PRO-3 to visualize cell nuclei. Some utricles were also labeled with fluorogenic caspase-3, -8, or -9 indicators to evaluate the mechanism of programmed cell death. Mefloquine treatment caused a dose-dependent loss of utricular hair cells. Treatment with 10 μM caused a slight reduction, 50 μM caused a significant reduction, and 200 μM destroyed nearly all the hair cells. Hair cell nuclei in mefloquine-treated utricles were condensed and fragmented, morphological features of apoptosis. Mefloquine-treated utricles were positive for the extrinsic initiator caspase-8 and intrinsic initiator caspase-9 and downstream executioner caspase-3. These results indicate that mefloquine can induce significant hair cell degeneration in the postnatal rat utricle and that mefloquine-induced hair cell death is initiated by both caspase-8 and caspase-9.

  11. The use of displacement damage dose to correlate degradation in solar cells exposed to different radiations

    NASA Technical Reports Server (NTRS)

    Summers, Geoffrey P.; Burke, Edward A.; Shapiro, Philip; Statler, Richard; Messenger, Scott R.; Walters, Robert J.

    1994-01-01

    It has been found useful in the past to use the concept of 'equivalent fluence' to compare the radiation response of different solar cell technologies. Results are usually given in terms of an equivalent 1 MeV electron or an equivalent 10 MeV proton fluence. To specify cell response in a complex space-radiation environment in terms of an equivalent fluence, it is necessary to measure damage coefficients for a number of representative electron and proton energies. However, at the last Photovoltaic Specialist Conference we showed that nonionizing energy loss (NIEL) could be used to correlate damage coefficients for protons, using measurements for GaAs as an example. This correlation means that damage coefficients for all proton energies except near threshold can be predicted from a measurement made at one particular energy. NIEL is the exact equivalent for displacement damage of linear energy transfer (LET) for ionization energy loss. The use of NIEL in this way leads naturally to the concept of 10 MeV equivalent proton fluence. The situation for electron damage is more complex, however. It is shown that the concept of 'displacement damage dose' gives a more general way of unifying damage coefficients. It follows that 1 MeV electron equivalent fluence is a special case of a more general quantity for unifying electron damage coefficients which we call the 'effective 1 MeV electron equivalent dose'.

  12. Role of Myo/Nog Cells in Neuroprotection: Evidence from the Light Damaged Retina.

    PubMed

    Brandli, Alice; Gerhart, Jacquelyn; Sutera, Christopher K; Purushothuman, Sivaraman; George-Weinstein, Mindy; Stone, Jonathan; Bravo-Nuevo, Arturo

    2017-01-01

    To identify Myo/Nog cells in the adult retina and test their role in protecting retinal photoreceptors from light damage. Light damage was induced by exposing albino rats raised in dim cyclic light to 1000 lux light for 24 hours. In one group of rats, Myo/Nog cells were purified from rat brain tissue by magnetic cell sorting following binding of the G8 monoclonal antibody (mAb). These cells were injected into the vitreous humour of the eye within 2 hours following bright light exposure. Retinal function was assessed using full-field, flash electroretinogram (ERG) before and after treatment. The numbers of Myo/Nog cells, apoptotic photoreceptors, and the expression of glial fibrillary acidic protein (GFAP) in Muller cells were assessed by immunohistochemistry. Myo/Nog cells were present in the undamaged retina in low numbers. Light induced damage increased their numbers, particularly in the choroid, ganglion cell layer and outer plexiform layer. Intravitreal injection of G8-positive (G8+) cells harvested from brain mitigated all the effects of light damage examined, i.e. loss of retinal function (ERG), death of photoreceptors and the stress-induced expression of GFAP in Muller cells. Some of the transplanted G8+ cells were integrated into the retina from the vitreous. Myo/Nog cells are a subpopulation of cells that are present in the adult retina. They increase in number in response to light induced stress. Intravitreal injection of Myo/Nog cells was protective to the retina, in part, by reducing retinal stress as measured by the Muller cell response. These results suggest that Myo/Nog cells, or the factors they produce, are neuroprotective and may be therapeutic in neurodegenerative retinal diseases.

  13. Antioxidant Enzymes Reduce DNA Damage and Early Activation of Valvular Interstitial Cells in Aortic Valve Sclerosis

    PubMed Central

    Branchetti, Emanuela; Sainger, Rachana; Poggio, Paolo; Grau, Juan B.; Patterson-Fortin, Jeffrey; Bavaria, Joseph E.; Chorny, Michael; Lai, Eric; Gorman, Robert C.; Levy, Robert J.; Ferrari, Giovanni

    2014-01-01

    Objective Accumulation of reactive oxygen species (ROS) and remodeling of the microstructure of the cusp characterize aortic valve sclerosis, the early phase of calcific aortic valve disease. These events are associated with activation of valvular interstitial cells (VICs) toward an osteogenic-like phenotype. Because ROS cause DNA damage and transcriptional activation we investigated the relationship between ROS, DNA damage response, and transdifferentiation of VICs. Methods and Results Human aortic valve cusps and patient-matched VICs were collected from 39 patients both with and without calcific aortic valve disease. VICs were exposed to hydrogen peroxide (0.1–1 mmol/L) after cell transduction with extracellular superoxide dismutase/catalase adenoviruses and characterized for DNA-damage response, osteogenic transdifferentiation, and calcification. ROS induce relocalization of phosphorylated γH2AX, MRE11, and XRCC1 proteins with expression of osteogenic signaling molecule RUNX2 via AKT. We report a sustained activation of γH2AX in aortic valve sclerosis-derived VICs suggesting their impaired ability to repair DNA damage. Adenovirus superoxide dismutase/catalase transduction decreases ROS-induced DNA damage and VIC transdifferentiation in aortic valve sclerosis-derived cells. Finally, adenoviral transduction with catalase reverts ROS-mediated calcification and cellular transdifferentiation. Conclusion We conclude that the ROS-induced DNA damage response is dysfunctional in early asymptomatic stages of calcific aortic valve disease. We unveiled an association among ROS, DNA-damage response, and cellular transdifferentiation, reversible by antioxidant enzymes delivery. PMID:23241403

  14. Ultrastructural study of mitochondrial damage in CHO cells exposed to hyperthermia.

    PubMed

    Cole, A; Armour, E P

    1988-09-01

    A unique direct-view stereo electron microscope technique was used to visualize the structure and three-dimensional distributions of mitochondria in CHO cells in situ following hyperthermic treatments. Aberrations induced by various heating regimens were recorded. The protocol included a trypsin digestion that may have enhanced the expression of the initial heat damage. The developed damage was observed as increasing levels of mitochondrial distortion, swelling, and dissociation. Minimal damage was induced at 42 degrees C for exposures of up to 4 h, while significant damage was induced at 43 degrees C for exposures of more than 30 min and at 45 degrees C for exposures of more than 10 min. For moderate exposures, a partial recovery of mitochondrial integrity was observed when the heat treatment was followed by incubation at 37 degrees C for 24 h. Mitochondrial damage was related to the heat dose in that increasing treatment temperature resulted in greater damage, but when compared to cell survival the damage did not parallel cell killing under all time-temperature conditions.

  15. Electron beam induced radiation damage in the catalyst layer of a proton exchange membrane fuel cell.

    PubMed

    He, Qianping; Chen, Jihua; Keffer, David J; Joy, David C

    2014-01-01

    Electron microscopy is an essential tool for the evaluation of microstructure and properties of the catalyst layer (CL) of proton exchange membrane fuel cells (PEMFCs). However, electron microscopy has one unavoidable drawback, which is radiation damage. Samples suffer temporary or permanent change of the surface or bulk structure under radiation damage, which can cause ambiguity in the characterization of the sample. To better understand the mechanism of radiation damage of CL samples and to be able to separate the morphological features intrinsic to the material from the consequences of electron radiation damage, a series of experiments based on high-angle annular dark-field-scanning transmission scanning microscope (HAADF-STEM), energy filtering transmission scanning microscope (EFTEM), and electron energy loss spectrum (EELS) are conducted. It is observed that for thin samples (0.3-1 times λ), increasing the incident beam energy can mitigate the radiation damage. Platinum nanoparticles in the CL sample facilitate the radiation damage. The radiation damage of the catalyst sample starts from the interface of Pt/C or defective thin edge and primarily occurs in the form of mass loss accompanied by atomic displacement and edge curl. These results provide important insights on the mechanism of CL radiation damage. Possible strategies of mitigating the radiation damage are provided. © 2013 Wiley Periodicals, Inc.

  16. Nanosecond pulsed electric field induced cytoskeleton, nuclear membrane and telomere damage adversely impact cell survival.

    PubMed

    Stacey, M; Fox, P; Buescher, S; Kolb, J

    2011-10-01

    We investigated the effects of nanosecond pulsed electric fields (nsPEF) on three human cell lines and demonstrated cell shrinkage, breakdown of the cytoskeleton, nuclear membrane and chromosomal telomere damage. There was a differential response between cell types coinciding with cell survival. Jurkat cells showed cytoskeleton, nuclear membrane and telomere damage that severely impacted cell survival compared to two adherent cell lines. Interestingly, disruption of the actin cytoskeleton in adherent cells prior to nsPEF exposure significantly reduced cell survival. We conclude that nsPEF applications are able to induce damage to the cytoskeleton and nuclear membrane. Telomere sequences, regions that tether and stabilize DNA to the nuclear membrane, are severely compromised as measured by a pan-telomere probe. Internal pore formation following nsPEF applications has been described as a factor in induced cell death. Here we suggest that nsPEF induced physical changes to the cell in addition to pore formation need to be considered as an alternative method of cell death. We suggest nsPEF electrochemical induced depolymerization of actin filaments may account for cytoskeleton and nuclear membrane anomalies leading to sensitization. Copyright © 2011 Elsevier B.V. All rights reserved.

  17. Human POLD1 modulates cell cycle progression and DNA damage repair.

    PubMed

    Song, Jing; Hong, Ping; Liu, Chengeng; Zhang, Yueqi; Wang, Jinling; Wang, Peichang

    2015-06-19

    The activity of eukaryotic DNA polymerase delta (Pol δ) plays an essential role in genome stability through its effects on DNA replication and repair. The p125 catalytic subunit of Pol δ is encoded by POLD1 gene in human cells. To clarify biological functions of POLD1, we investigated the effects of POLD1 overexpression or downregulation on cell proliferation, cell cycle progression, DNA synthesis and oxidative DNA damage induced by H2O2. HEK293 cells were transfected with POLD1 expression plasmid or shRNA, cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells were analyzed. HEK293 cells were transfected with POLD1 expression plasmid or shRNA. POLD1 downregulation by shRNA suppressed cell proliferation, cell cycle progression, and DNA synthesis in HEK293 cells. However, POLD1 overexpression had no significant effects on these processes. Finally, comet assay showed that POLD1 downregulation led to increased DNA damage. Our results suggest that human POLD1 plays important role in the regulation of cell cycle progression and DNA damage repair.

  18. The DNA damage/repair cascade in glioblastoma cell lines after chemotherapeutic agent treatment

    PubMed Central

    ANNOVAZZI, LAURA; CALDERA, VALENTINA; MELLAI, MARTA; RIGANTI, CHIARA; BATTAGLIA, LUIGI; CHIRIO, DANIELA; MELCARNE, ANTONIO; SCHIFFER, DAVIDE

    2015-01-01

    Therapeutic resistance in glioblastoma multiforme (GBM) has been linked to a subpopulation of cells with stem cell-like properties, the glioma stem cells (GSCs), responsible for cancer progression and recurrence. This study investigated the in vitro cytotoxicity of three chemotherapeutics, temozolomide (TMZ), doxorubicin (Dox) and paclitaxel (PTX) on glioma cell lines, by analyzing the molecular mechanisms leading to DNA repair and cell resistance, or to cell death. The drugs were tested on 16 GBM cell lines, grown as neurospheres (NS) or adherent cells (AC), by studying DNA damage occurrence by Comet assay, the expression by immunofluorescence and western blotting of checkpoint/repair molecules and apoptosis. The three drugs were able to provoke a genotoxic injury and to inhibit dose- and time-dependently cell proliferation, more evidently in AC than in NS. The first cell response to DNA damage was the activation of the damage sensors (p-ATM, p-53BP1, γ-H2AX), followed by repair effectors; the expression of checkpoint/repair molecules appeared higher in NS than in AC. The non-homologous repair pathway (NHEJ) seemed more involved than the homologous one (HR). Apoptosis occurred after long treatment times, but only a small percentage of cells in NS underwent death, even at high drug concentration, whereas most cells survived in a quiescent state and resumed proliferation after drug removal. In tumor specimens, checkpoint/repair proteins were constitutively expressed in GBMs, but not in low-grade gliomas. PMID:25892134

  19. Cell survival, DNA damage, and oncogenic transformation after a transient and reversible apoptotic response

    PubMed Central

    Tang, Ho Lam; Tang, Ho Man; Mak, Keng Hou; Hu, Shaomin; Wang, Shan Shan; Wong, Kit Man; Wong, Chung Sing Timothy; Wu, Hoi Yan; Law, Hiu Tung; Liu, Kan; Talbot, C. Conover; Lau, Wan Keung; Montell, Denise J.; Fung, Ming Chiu

    2012-01-01

    Apoptosis serves as a protective mechanism by eliminating damaged cells through programmed cell death. After apoptotic cells pass critical checkpoints, including mitochondrial fragmentation, executioner caspase activation, and DNA damage, it is assumed that cell death inevitably follows. However, this assumption has not been tested directly. Here we report an unexpected reversal of late-stage apoptosis in primary liver and heart cells, macrophages, NIH 3T3 fibroblasts, cervical cancer HeLa cells, and brain cells. After exposure to an inducer of apoptosis, cells exhibited multiple morphological and biochemical hallmarks of late-stage apoptosis, including mitochondrial fragmentation, caspase-3 activation, and DNA damage. Surprisingly, the vast majority of dying cells arrested the apoptotic process and recovered when the inducer was washed away. Of importance, some cells acquired permanent genetic changes and underwent oncogenic transformation at a higher frequency than controls. Global gene expression analysis identified a molecular signature of the reversal process. We propose that reversal of apoptosis is an unanticipated mechanism to rescue cells from crisis and propose to name this mechanism “anastasis” (Greek for “rising to life”). Whereas carcinogenesis represents a harmful side effect, potential benefits of anastasis could include preservation of cells that are difficult to replace and stress-induced genetic diversity. PMID:22535522

  20. Overtraining is associated with DNA damage in blood and skeletal muscle cells of Swiss mice

    PubMed Central

    2013-01-01

    Background The alkaline version of the single-cell gel (comet) assay is a useful method for quantifying DNA damage. Although some studies on chronic and acute effects of exercise on DNA damage measured by the comet assay have been performed, it is unknown if an aerobic training protocol with intensity, volume, and load clearly defined will improve performance without leading to peripheral blood cell DNA damage. In addition, the effects of overtraining on DNA damage are unknown. Therefore, this study aimed to examine the effects of aerobic training and overtraining on DNA damage in peripheral blood and skeletal muscle cells in Swiss mice. To examine possible changes in these parameters with oxidative stress, we measured reduced glutathione (GSH) levels in total blood, and GSH levels and lipid peroxidation in muscle samples. Results Performance evaluations (i.e., incremental load and exhaustive tests) showed significant intra and inter-group differences. The overtrained (OTR) group showed a significant increase in the percentage of DNA in the tail compared with the control (C) and trained (TR) groups. GSH levels were significantly lower in the OTR group than in the C and TR groups. The OTR group had significantly higher lipid peroxidation levels compared with the C and TR groups. Conclusions Aerobic and anaerobic performance parameters can be improved in training at maximal lactate steady state during 8 weeks without leading to DNA damage in peripheral blood and skeletal muscle cells or to oxidative stress in skeletal muscle cells. However, overtraining induced by downhill running training sessions is associated with DNA damage in peripheral blood and skeletal muscle cells, and with oxidative stress in skeletal muscle cells and total blood. PMID:24099482

  1. DDR-mediated crosstalk between DNA-damaged cells and their microenvironment

    PubMed Central

    Malaquin, Nicolas; Carrier-Leclerc, Audrey; Dessureault, Mireille; Rodier, Francis

    2015-01-01

    The DNA damage response (DDR) is an evolutionarily conserved signaling cascade that senses and responds to double-strand DNA breaks by organizing downstream cellular events, ranging from appropriate DNA repair to cell cycle checkpoints. In higher organisms, the DDR prevents neoplastic transformation by directly protecting the information contained in the genome and by regulating cell fate decisions, like apoptosis and senescence, to ensure the removal of severely damaged cells. In addition to these well-studied cell-autonomous effects, emerging evidence now shows that the DDR signaling cascade can also function in a paracrine manner, thus influencing the biology of the surrounding cellular microenvironment. In this context, the DDR plays an emerging role in shaping the damaged tumor microenvironment through the regulation of tissue repair and local immune responses, thereby providing a promising avenue for novel therapeutic interventions. Additionally, while DDR-mediated extracellular signals can convey information to surrounding, undamaged cells, they can also feedback onto DNA-damaged cells to reinforce selected signaling pathways. Overall, these extracellular DDR signals can be subdivided into two time-specific waves: a rapid bystander effect occurring within a few hours of DNA damage; and a late, delayed, senescence-associated secretory phenotype generally requiring multiple days to establish. Here, we highlight and discuss examples of rapid and late DDR–mediated extracellular alarm signals. PMID:25815006

  2. DDR-mediated crosstalk between DNA-damaged cells and their microenvironment.

    PubMed

    Malaquin, Nicolas; Carrier-Leclerc, Audrey; Dessureault, Mireille; Rodier, Francis

    2015-01-01

    The DNA damage response (DDR) is an evolutionarily conserved signaling cascade that senses and responds to double-strand DNA breaks by organizing downstream cellular events, ranging from appropriate DNA repair to cell cycle checkpoints. In higher organisms, the DDR prevents neoplastic transformation by directly protecting the information contained in the genome and by regulating cell fate decisions, like apoptosis and senescence, to ensure the removal of severely damaged cells. In addition to these well-studied cell-autonomous effects, emerging evidence now shows that the DDR signaling cascade can also function in a paracrine manner, thus influencing the biology of the surrounding cellular microenvironment. In this context, the DDR plays an emerging role in shaping the damaged tumor microenvironment through the regulation of tissue repair and local immune responses, thereby providing a promising avenue for novel therapeutic interventions. Additionally, while DDR-mediated extracellular signals can convey information to surrounding, undamaged cells, they can also feedback onto DNA-damaged cells to reinforce selected signaling pathways. Overall, these extracellular DDR signals can be subdivided into two time-specific waves: a rapid bystander effect occurring within a few hours of DNA damage; and a late, delayed, senescence-associated secretory phenotype generally requiring multiple days to establish. Here, we highlight and discuss examples of rapid and late DDR-mediated extracellular alarm signals.

  3. Acute hydrodynamic damage induced by SPLITT fractionation and centrifugation in red blood cells.

    PubMed

    Urbina, Adriana; Godoy-Silva, Ruben; Hoyos, Mauricio; Camacho, Marcela

    2016-05-01

    Though blood bank processing traditionally employs centrifugation, new separation techniques may be appealing for large scale processes. Split-flow fractionation (SPLITT) is a family of techniques that separates in absence of labelling and uses very low flow rates and force fields, and is therefore expected to minimize cell damage. However, the hydrodynamic stress and possible consequent damaging effects of SPLITT fractionation have not been yet examined. The aim of this study was to investigate the hydrodynamic damage of SPLITT fractionation to human red blood cells, and to compare these effects with those induced by centrifugation. Peripheral whole blood samples were collected from healthy volunteers. Samples were diluted in a buffered saline solution, and were exposed to SPLITT fractionation (flow rates 1-10 ml/min) or centrifugation (100-1500 g) for 10 min. Cell viability, shape, diameter, mean corpuscular hemoglobin, and membrane potential were measured. Under the operating conditions employed, both SPLITT and centrifugation maintained cell viability above 98%, but resulted in significant sublethal damage, including echinocyte formation, decreased cell diameter, decreased mean corpuscular hemoglobin, and membrane hyperpolarization which was inhibited by EGTA. Wall shear stress and maximum energy dissipation rate showed significant correlation with lethal and sublethal damage. Our data do not support the assumption that SPLITT fractionation induces very low shear stress and is innocuous to cell function. Some changes in SPLITT channel design are suggested to minimize cell damage. Measurement of membrane potential and cell diameter could provide a new, reliable and convenient basis for evaluation of hydrodynamic effects on different cell models, allowing identification of optimal operating conditions on different scales. Copyright © 2016 Elsevier B.V. All rights reserved.

  4. Periodic annealing of radiation damage in GaAs solar cells

    NASA Technical Reports Server (NTRS)

    Loo, R. Y.; Knechtli, R. C.; Kamath, G. S.

    1980-01-01

    Continuous annealing of GaAs solar cells is compared with periodic annealing to determine their relative effectiveness in minimizing proton radiation damage. It is concluded that continuous annealing of the cells in space at 150 C can effectively reduce the proton radiation damage to the GaAs solar cells. Periodic annealing is most effective if it can be initiated at relatively low fluences (approximating continuous annealing), especially if low temperatures of less than 200 C are to be used. If annealing is started only after the fluence of the damaging protons has accumulated to a high value 10 to the 11th power sq/pcm), effective annealing is still possible at relatively high temperatures. Finally, since electron radiation damage anneals even more easily than proton radiation damage, substantial improvements in GaAs solar cell life can be achieved by incorporating the proper annealing capabilities in solar panels for practical space missions where both electron and proton radiation damage have to be minimized.

  5. The effect of a DNA damaging agent on embryonic cell cycles of the cnidarian Hydractinia echinata.

    PubMed

    Su, Tin Tin

    2010-07-23

    The onset of gastrulation at the Mid-Blastula Transition can accompany profound changes in embryonic cell cycles including the introduction of gap phases and the transition from maternal to zygotic control. Studies in Xenopus and Drosophila embryos have also found that cell cycles respond to DNA damage differently before and after MBT (or its equivalent, MZT, in Drosophila). DNA checkpoints are absent in Xenopus cleavage cycles but are acquired during MBT. Drosophila cleavage nuclei enter an abortive mitosis in the presence of DNA damage whereas post-MZT cells delay the entry into mitosis. Despite attributes that render them workhorses of embryonic cell cycle studies, Xenopus and Drosophila are hardly representative of diverse animal forms that exist. To investigate developmental changes in DNA damage responses in a distant phylum, I studied the effect of an alkylating agent, Methyl Methanesulfonate (MMS), on embryos of Hydractinia echinata. Hydractinia embryos are found to differ from Xenopus embryos in the ability to respond to a DNA damaging agent in early cleavage but are similar to Xenopus and Drosophila embryos in acquiring stronger DNA damage responses and greater resistance to killing by MMS after the onset of gastrulation. This represents the first study of DNA damage responses in the phylum Cnidaria.

  6. Parainfluenza Virus Infection Sensitizes Cancer Cells to DNA-Damaging Agents: Implications for Oncolytic Virus Therapy.

    PubMed

    Fox, Candace R; Parks, Griffith D

    2018-04-01

    A parainfluenza virus 5 (PIV5) with mutations in the P/V gene (P/V-CPI - ) is restricted for spread in normal cells but not in cancer cells in vitro and is effective at reducing tumor burdens in mouse model systems. Here we show that P/V-CPI - infection of HEp-2 human laryngeal cancer cells results in the majority of the cells dying, but unexpectedly, over time, there is an emergence of a population of cells that survive as P/V-CPI - persistently infected (PI) cells. P/V-CPI - PI cells had elevated levels of basal caspase activation, and viability was highly dependent on the activity of cellular inhibitor-of-apoptosis proteins (IAPs) such as Survivin and XIAP. In challenge experiments with external inducers of apoptosis, PI cells were more sensitive to cisplatin-induced DNA damage and cell death. This increased cisplatin sensitivity correlated with defects in DNA damage signaling pathways such as phosphorylation of Chk1 and translocation of damage-specific DNA binding protein 1 (DDB1) to the nucleus. Cisplatin-induced killing of PI cells was sensitive to the inhibition of wild-type (WT) p53-inducible protein 1 (WIP1), a phosphatase which acts to terminate DNA damage signaling pathways. A similar sensitivity to cisplatin was seen with cells during acute infection with P/V-CPI - as well as during acute infections with WT PIV5 and the related virus human parainfluenza virus type 2 (hPIV2). Our results have general implications for the design of safer paramyxovirus-based vectors that cannot establish PI as well as the potential for combining chemotherapy with oncolytic RNA virus vectors. IMPORTANCE There is intense interest in developing oncolytic viral vectors with increased potency against cancer cells, particularly those cancer cells that have gained resistance to chemotherapies. We have found that infection with cytoplasmically replicating parainfluenza virus can result in increases in the killing of cancer cells by agents that induce DNA damage, and this is linked

  7. Quinacrine pretreatment reduces microwave-induced neuronal damage by stabilizing the cell membrane

    PubMed Central

    Ding, Xue-feng; Wu, Yan; Qu, Wen-rui; Fan, Ming; Zhao, Yong-qi

    2018-01-01

    Quinacrine, widely used to treat parasitic diseases, binds to cell membranes. We previously found that quinacrine pretreatment reduced microwave radiation damage in rat hippocampal neurons, but the molecular mechanism remains poorly understood. Considering the thermal effects of microwave radiation and the protective effects of quinacrine on heat damage in cells, we hypothesized that quinacrine would prevent microwave radiation damage to cells in a mechanism associated with cell membrane stability. To test this, we used retinoic acid to induce PC12 cells to differentiate into neuron-like cells. We then pretreated the neurons with quinacrine (20 and 40 mM) and irradiated them with 50 mW/cm2 microwaves for 3 or 6 hours. Flow cytometry, atomic force microscopy and western blot assays revealed that irradiated cells pretreated with quinacrine showed markedly less apoptosis, necrosis, and membrane damage, and greater expression of heat shock protein 70, than cells exposed to microwave irradiation alone. These results suggest that quinacrine stabilizes the neuronal membrane structure by upregulating the expression of heat shock protein 70, thus reducing neuronal injury caused by microwave radiation. PMID:29623929

  8. DNA damage and decisions: CtIP coordinates DNA repair and cell cycle checkpoints.

    PubMed

    You, Zhongsheng; Bailis, Julie M

    2010-07-01

    Maintenance of genome stability depends on efficient, accurate repair of DNA damage. DNA double-strand breaks (DSBs) are among the most lethal types of DNA damage, with the potential to cause mutation, chromosomal rearrangement, and genomic instability that could contribute to cancer. DSB damage can be repaired by various pathways including nonhomologous end-joining (NHEJ) and homologous recombination (HR). However, the cellular mechanisms that regulate the choice of repair pathway are not well understood. Recent studies suggest that the tumor suppressor protein CtIP controls the decision to repair DSB damage by HR. It does so by regulating the initiation of DSB end resection after integrating signals from the DNA damage checkpoint response and cell cycle cues. Copyright 2010 Elsevier Ltd. All rights reserved.

  9. [Role of cytoglobin in protecting hepatic stellate cells against oxidation induced damage].

    PubMed

    Lü, Yinghui; Wang, Qizhao; Li, Zhaofa; Diao, Yong; Xu, Rui'an

    2011-08-01

    The aim of this study was to reveal the protection role and the related mechanism of cytoglobin on the oxidation induced hepatic stellate cell damage. We applied siRNA to interfere the endogenous cytoglobin gene, used recombinant cytoglobin protein to treat the completely activated human hepatic stellate cell line LX-2 and the incompletely activated primary rat hepatic stellate cells, or over-expressed cytoglobin protein in LX-2 cells. We used two different oxidative-stress related models, the hydrogen peroxide model and the iron-overload model in our experiments and investigated the proliferation status and the intracellular superoxide level of the cells. The results showed that endogenous cytoglobin exerted significant protective effects on hydrogen peroxide or iron-overload induced LX-2 cell damage, confirming that upregulation of cytoglobin was the protective response of activated hepatic stellate cells to oxidative stress. Recombinant cytoglobin protein could protect LX-2 cells from oxidation induced damage, and prevent primary rat hepatic stellate cells from excessive proliferation and injury. The cytoplasmic reactive oxygen species (ROS) scavenging capacity of the recombinant cytoglobin protein was not as good as its capacity in scavenging ROS outside the cells, likely owing to the lack of active transporting mechanisms. Intracellular over-expression of cytoglobin protein could exert significant protective effect on LX-2 cells treated with hydrogen peroxide or iron-overload. Our results would accelerate the exploitation of new anti-fibrotic targets.

  10. Gramicidin A induces metabolic dysfunction and energy depletion leading to cell death in renal cell carcinoma cells.

    PubMed

    David, Justin M; Owens, Tori A; Barwe, Sonali P; Rajasekaran, Ayyappan K

    2013-11-01

    Ionophores are lipid-soluble organic molecules that disrupt cellular transmembrane potential by rendering biologic membranes permeable to specific ions. They include mobile-carriers that complex with metal cations and channel-formers that insert into the membrane to form hydrophilic pores. Although mobile-carriers possess anticancer properties, investigations on channel-formers are limited. Here, we used the channel-forming ionophore gramicidin A to study its effects on the growth and survival of renal cell carcinoma (RCC) cells. RCC is a histologically heterogeneous malignancy that is highly resistant to conventional treatments. We found that gramicidin A reduced the in vitro viability of several RCC cell lines at submicromolar concentrations (all IC50 < 1.0 μmol/L). Gramicidin A exhibited similar toxicity in RCC cells regardless of histologic subtype or the expression of either the von Hippel-Lindau tumor suppressor gene or its downstream target, hypoxia-inducible factor-1α. Gramicidin A decreased cell viability equal to or greater than the mobile-carrier monensin depending on the cell line. Mechanistic examination revealed that gramicidin A blocks ATP generation by inhibiting oxidative phosphorylation and glycolysis, leading to cellular energy depletion and nonapoptotic cell death. Finally, gramicidin A effectively reduced the growth of RCC tumor xenografts in vivo. These results show a novel application of gramicidin A as a potential therapeutic agent for RCC therapy. ©2013 AACR.

  11. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, affects on the cellular response to DNA damage induced by exposures to radiation or other toxic chemicals will have an impact on the radiation risks for the astronauts, as well as on the mutation rate in microorganisms, is still an open question. Although the possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate the effects of spaceflight on the cellular response to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induces DNA damages including the double strand breaks (DSB) similar to the ionizing radiation. Damage in the DNA was measured by the phosphorylation of a histone protein H2AX (-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in the DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ti-67 signals. Our results suggested that the difference in -H2AX between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect the response of the DNA damage response genes to bleomycin treatment.

  12. Cellular Response to Bleomycin-Induced DNA Damage in Human Fibroblast Cells in Space

    NASA Technical Reports Server (NTRS)

    Lu, Tao; Zhang, Ye; Wong, Michael; Stodieck, Louis; Karouia, Fathi; Wu, Honglu

    2015-01-01

    Outside the protection of the geomagnetic field, astronauts and other living organisms are constantly exposed to space radiation that consists of energetic protons and other heavier charged particles. Whether spaceflight factors, microgravity in particular, have effects on cellular responses to DNA damage induced by exposure to radiation or cytotoxic chemicals is still unknown, as is their impact on the radiation risks for astronauts and on the mutation rate in microorganisms. Although possible synergistic effects of space radiation and other spaceflight factors have been investigated since the early days of the human space program, the published results were mostly conflicting and inconsistent. To investigate effects of spaceflight on cellular responses to DNA damages, human fibroblast cells flown to the International Space Station (ISS) were treated with bleomycin for three hours in the true microgravity environment, which induced DNA damages including double-strand breaks (DSB) similar to the ionizing radiation. Damages in the DNA were measured by the phosphorylation of a histone protein H2AX (g-H2AX), which showed slightly more foci in the cells on ISS than in the ground control. The expression of genes involved in DNA damage response was also analyzed using the PCR array. Although a number of the genes, including CDKN1A and PCNA, were significantly altered in the cells after bleomycin treatment, no significant difference in the expression profile of DNA damage response genes was found between the flight and ground samples. At the time of the bleomycin treatment, the cells on the ISS were found to be proliferating faster than the ground control as measured by the percentage of cells containing positive Ki-67 signals. Our results suggested that the difference in g-H2AX focus counts between flight and ground was due to the faster growth rate of the cells in space, but spaceflight did not affect initial transcriptional responses of the DNA damage response genes to

  13. Iron intake, red cell indicators of iron status, and DNA damage in young subjects.

    PubMed

    Prá, Daniel; Bortoluzzi, Angelica; Müller, Luiza Louzada; Hermes, Liziane; Horta, Jorge André; Maluf, Sharbel Weidner; Henriques, João Antonio Pêgas; Fenech, Michael; Franke, Silvia Isabel Rech

    2011-03-01

    This study evaluated the association between primary DNA damage and chromosomal damage with iron intake and red blood cell parameters of iron status in a sample of healthy children and adolescents from a low-socioeconomic community. The level of primary DNA damage was assessed using an alkaline comet assay and the level of chromosomal damage was assessed using the cytokinesis-block micronucleus assay. A automated complete blood count was used to evaluate red blood cell status. The intake of iron was measured using a food-recall questionnaire. According to hemoglobin levels, only 1 of the 30 subjects evaluated was anemic. Nevertheless, 43% of the sampled subjects showed decreased mean corpuscular volume in addition to an increased amount of primary DNA damage (P < 0.05). Mean corpuscular volume was negatively correlated with primary DNA damage (r = -0.429, P = 0.020) but not with chromosomal damage. The association between iron and primary DNA damage showed a U-shaped curve, indicating that an intake of approximately 15 mg of iron per day (up to two-fold of the dietary recommended intake) could minimize primary DNA damage in this age group. The frequency of micronuclei and nucleoplasmic bridges, indicators of chromosomal breakage/loss and chromosomal end-fusions, respectively, showed a negative correlation with iron intake. These results indicate that an intake of iron >15 mg/d could increase genomic stability in binucleated lymphocytes of the same group. An intake of iron ≥ 15 mg/d can decrease DNA damage in young subjects. Copyright © 2011 Elsevier Inc. All rights reserved.

  14. Mouse Bone Marrow-Derived Endothelial Progenitor Cells Do Not Restore Radiation-Induced Microvascular Damage

    PubMed Central

    Seemann, Ingar; te Poele, Johannes A. M.; Hoving, Saske; Stewart, Fiona A.

    2014-01-01

    Background. Radiotherapy is commonly used to treat breast and thoracic cancers but it also causes delayed microvascular damage and increases the risk of cardiac mortality. Endothelial cell proliferation and revascularization are crucial to restore microvasculature damage and maintain function of the irradiated heart. We have therefore examined the potential of bone marrow-derived endothelial progenitor cells (BM-derived EPCs) for restoration of radiation-induced microvascular damage. Material & Methods. 16 Gy was delivered to the heart of adult C57BL/6 mice. Mice were injected with BM-derived EPCs, obtained from Eng+/+ or Eng+/− mice, 16 weeks and 28 weeks after irradiation. Morphological damage was evaluated at 40 weeks in transplanted mice, relative to radiation only and age-matched controls. Results. Cardiac irradiation decreased microvascular density and increased endothelial damage in surviving capillaries (decrease alkaline phosphatase expression and increased von Willebrand factor). Microvascular damage was not diminished by treatment with BM-derived EPCs. However, BM-derived EPCs from both Eng+/+ and Eng+/− mice diminished radiation-induced collagen deposition. Conclusion. Treatment with BM-derived EPCs did not restore radiation-induced microvascular damage but it did inhibit fibrosis. Endoglin deficiency did not impair this process. PMID:25101181

  15. Nek1 silencing slows down DNA repair and blocks DNA damage-induced cell cycle arrest.

    PubMed

    Pelegrini, Alessandra Luíza; Moura, Dinara Jaqueline; Brenner, Bethânia Luise; Ledur, Pitia Flores; Maques, Gabriela Porto; Henriques, João Antônio Pegas; Saffi, Jenifer; Lenz, Guido

    2010-09-01

    Never in mitosis A (NIMA)-related kinases (Nek) are evolutionarily conserved proteins structurally related to the Aspergillus nidulans mitotic regulator NIMA. Nek1 is one of the 11 isoforms of the Neks identified in mammals. Different lines of evidence suggest the participation of Nek1 in response to DNA damage, which is also supported by the interaction of this kinase with proteins involved in DNA repair pathways and cell cycle regulation. In this report, we show that cells with Nek1 knockdown (KD) through stable RNA interference present a delay in DNA repair when treated with methyl-methanesulfonate (MMS), hydrogen peroxide (H(2)O(2)) and cisplatin (CPT). In particular, interstrand cross links induced by CPT take much longer to be resolved in Nek1 KD cells when compared to wild-type (WT) cells. In KD cells, phosphorylation of Chk1 in response to CPT was strongly reduced. While WT cells accumulate in G(2)/M after DNA damage with MMS and H(2)O(2), Nek1 KD cells do not arrest, suggesting that G(2)/M arrest induced by the DNA damage requires Nek1. Surprisingly, CPT-treated Nek1 KD cells arrest with a 4N DNA content similar to WT cells. This deregulation in cell cycle control in Nek1 KD cells leads to an increased sensitivity to genotoxic agents when compared to WT cells. These results suggest that Nek1 is involved in the beginning of the cellular response to genotoxic stress and plays an important role in preventing cell death induced by DNA damage.

  16. Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

    NASA Astrophysics Data System (ADS)

    Unachukwu, Uchenna John; Warren, Alice; Li, Ze; Mishra, Shawn; Zhou, Jing; Sauane, Moira; Lim, Hyungsik; Vazquez, Maribel; Redenti, Stephen

    2016-03-01

    To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α - CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment.

  17. Cytoprotective effect of phloroglucinol on oxidative stress induced cell damage via catalase activation.

    PubMed

    Kang, Kyoung Ah; Lee, Kyoung Hwa; Chae, Sungwook; Zhang, Rui; Jung, Myung Sun; Ham, Young Min; Baik, Jong Seok; Lee, Nam Ho; Hyun, Jin Won

    2006-02-15

    We investigated the cytoprotective effect of phloroglucinol, which was isolated from Ecklonia cava (brown alga), against oxidative stress induced cell damage in Chinese hamster lung fibroblast (V79-4) cells. Phloroglucinol was found to scavenge 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical, hydrogen peroxide (H(2)O(2)), hydroxy radical, intracellular reactive oxygen species (ROS), and thus prevented lipid peroxidation. As a result, phloroglucinol reduced H(2)O(2) induced apoptotic cells formation in V79-4 cells. In addition, phloroglucinol inhibited cell damage induced by serum starvation and radiation through scavenging ROS. Phloroglucinol increased the catalase activity and its protein expression. In addition, catalase inhibitor abolished the protective effect of phloroglucinol from H(2)O(2) induced cell damage. Furthermore, phloroglucinol increased phosphorylation of extracellular signal regulated kinase (ERK). Taken together, the results suggest that phloroglucinol protects V79-4 cells against oxidative damage by enhancing the cellular catalase activity and modulating ERK signal pathway. (c) 2005 Wiley-Liss, Inc.

  18. Predicted molecular signaling guiding photoreceptor cell migration following transplantation into damaged retina

    PubMed Central

    Unachukwu, Uchenna John; Warren, Alice; Li, Ze; Mishra, Shawn; Zhou, Jing; Sauane, Moira; Lim, Hyungsik; Vazquez, Maribel; Redenti, Stephen

    2016-01-01

    To replace photoreceptors lost to disease or trauma and restore vision, laboratories around the world are investigating photoreceptor replacement strategies using subretinal transplantation of photoreceptor precursor cells (PPCs) and retinal progenitor cells (RPCs). Significant obstacles to advancement of photoreceptor cell-replacement include low migration rates of transplanted cells into host retina and an absence of data describing chemotactic signaling guiding migration of transplanted cells in the damaged retinal microenvironment. To elucidate chemotactic signaling guiding transplanted cell migration, bioinformatics modeling of PPC transplantation into light-damaged retina was performed. The bioinformatics modeling analyzed whole-genome expression data and matched PPC chemotactic cell-surface receptors to cognate ligands expressed in the light-damaged retinal microenvironment. A library of significantly predicted chemotactic ligand-receptor pairs, as well as downstream signaling networks was generated. PPC and RPC migration in microfluidic ligand gradients were analyzed using a highly predicted ligand-receptor pair, SDF-1α – CXCR4, and both PPCs and RPCs exhibited significant chemotaxis. This work present a systems level model and begins to elucidate molecular mechanisms involved in PPC and RPC migration within the damaged retinal microenvironment. PMID:26935401

  19. Correlating measured transient temperature rises with damage rate processes in cultured cells

    NASA Astrophysics Data System (ADS)

    Denton, Michael L.; Tijerina, Amanda J.; Gonzalez, Cherry C.; Gamboa, B. Giovana; Noojin, Gary D.; Ahmed, Elharith M.; Rickman, John M.; Dyer, Phillip H.; Rockwell, Benjamin A.

    2017-02-01

    Thermal damage rate processes in biological tissues are usually characterized by a kinetics approach. This stems from experimental data that show how the transformation of a specified biological property of cells or biomolecule (plating efficiency for viability, change in birefringence, tensile strength, etc.) is dependent upon both time and temperature. Here, two disparate approaches were used to study thermal damage rate processes in cultured retinal pigment epithelial cells. Laser exposure (photothermal) parameters included 2-μm laser exposure of non-pigmented cells and 532-nm exposures of cells possessing a variety of melanosome particle densities. Photothermal experiments used a mid-IR camera to record temperature histories with spatial resolution of about 8 μm, while fluorescence microscopy of the cell monolayers identified threshold damage at the boundary between live and dead cells. Photothermal exposure durations ranged from 0.05-20 s, and the effects of varying ambient temperature were investigated. Temperature during heat transfer using a water-jacketed cuvette was recorded with a fast microthermister, while damage and viability of the suspended cells were determined as percentages. Exposure durations for the heat transfer experiments ranged from 50- 60 s. Empirically-determined kinetic parameters for the two heating methods were compared with each other, and with values found in the literature.

  20. Blue light-activated hypocrellin B damages ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Jiang, Y.; Leung, A. W. N.; Xiang, J. Y.; Xu, C. S.

    2011-10-01

    In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hyppcrellin B concentration was kept at 2.5 μM and light doses from 0.5-4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

  1. Blue light-activated hypocrellin B damages ovarian cancer cells

    NASA Astrophysics Data System (ADS)

    Jiang, Y.; Leung, A. W. N.; Xiang, J. Y.; Xu, C. S.

    2012-01-01

    In the present study, a novel blue light source from LED was used to activate hypocrellin B in ovarian cancer HO-8910 cells. Hyppcrellin B concentration was kept at 2.5 μM and light doses from 0.5-4.0 J/cm2. Photocytotoxicity was investigated using MTT reduction assay and light microscopy after light irradiation. Cellular morphology was observed using transmission electron microscopy (TEM). MTT assay showed that the cytotoxicity of blue light-activated hypocrellin B in HO-8910 cells increased along with light dose. The observations from light microscopy reinforced the above results. TEM showed that microvillin disappearance, vacuole formation, chromatin condensation, and topical apoptotic body were observed in the cells treated by both light and hypocrellin B. The findings demonstrated that blue light from LED source could effectively activate hypocrellin B to cause the destruction of HO-8910 cells, indicating that Blue light-activated hypocrellin B might be potential therapeutic strategy in the management of ovarian cancer.

  2. NASA's high efficiency and radiation damage solar cell program

    NASA Technical Reports Server (NTRS)

    Randolph, L. P.

    1980-01-01

    The conversion efficiency and the life expectancy of solar cells and arrays were evaluated for space applications. Efforts were made to improve the understanding of the conversion of electromagnetic radiation to useful forms of energy. A broad range of advanced concepts were evaluated.

  3. The aggregation and inheritance of damaged proteins determines cell fate during mitosis

    PubMed Central

    Bufalino, Mary Rose; van der Kooy, Derek

    2014-01-01

    Recent evidence suggests that proliferating cells polarize damaged proteins during mitosis to protect one cell from aging, and that the structural conformation of damaged proteins mediates their toxicity. We report that the growth, resistance to stress, and differentiation characteristics of a cancer cell line (PC12) with an inducible Huntingtin (Htt) fused to enhanced green fluorescent protein (GFP) are dependent on the conformation of Htt. Cell progeny containing inclusion bodies have a longer cell cycle and increased resistance to stress than those with diffuse Htt. Using live imaging, we demonstrate that asymmetric division resulting from a cell containing a single inclusion body produces sister cells with different fates. The cell that receives the inclusion body has decreased proliferation and increased differentiation compared with its sister cell without Htt. This is the first report that reveals a functional consequence of the asymmetric division of damaged proteins in mammalian cells, and we suggest that this is a result of inclusion body-induced proteasome impairment. PMID:24553116

  4. In vitro DNA-damaging effects of intestinal and related tetrapyrroles in human cancer cells.

    PubMed

    Mölzer, Christine; Pfleger, Barbara; Putz, Elisabeth; Roßmann, Antonia; Schwarz, Ursula; Wallner, Marlies; Bulmer, Andrew C; Wagner, Karl-Heinz

    2013-02-15

    Epidemiological studies report a negative association between circulating bilirubin concentrations and the risk for cancer and cardiovascular disease. Structurally related tetrapyrroles also possess in vitro anti-genotoxic activity and may prevent mutation prior to malignancy. Furthermore, few data suggest that tetrapyrroles exert anti-carcinogenic effects via induction of cell cycle arrest and apoptosis. To further investigate whether tetrapyrroles provoke DNA-damage in human cancer cells, they were tested in the single cell gel electrophoresis assay (SCGE). Eight tetrapyrroles (unconjugated bilirubin, bilirubin ditaurate, biliverdin, biliverdin-/bilirubin dimethyl ester, urobilin, stercobilin and protoporphyrin) were added to cultured Caco2 and HepG2 cells and their effects on comet formation (% tail DNA) were assessed. Flow cytometric assessment (apoptosis/necrosis, cell cycle, intracellular radical species generation) assisted in revealing underlying mechanisms of intracellular action. Cells were incubated with tetrapyrroles at concentrations of 0.5, 5 and 17μM for 24h. Addition of 300μM tertiary-butyl hydroperoxide to cells served as a positive control. Tetrapyrrole incubation mostly resulted in increased DNA-damage (comet formation) in Caco2 and HepG2 cells. Tetrapyrroles that are concentrated within the intestine, including protoporphyrin, urobilin and stercobilin, led to significant comet formation in both cell lines, implicating the compounds in inducing DNA-damage and apoptosis in cancer cells found within organs of the digestive system. Copyright © 2012 Elsevier Inc. All rights reserved.

  5. [Elimination and excretion of adenylate kinases following cell damage].

    PubMed

    Sachsenheimer, W; Goody, R S; Schirmer, R H

    1975-07-01

    Adenylate kinases, small organ-specific isoenzymes which appear after tissue damage in the blood plasma are partly eliminated via the kidney. After intravenous administration of 3000 enzyme units of 14C-labelled adenylate kinase to rats, about 50% of the enzyme and of the radioactivity are found in the urine within 7 minutes. The elimination of adenylate kinase from the serum occurs in two phases, a faster (half-life 16 minutes) and a slower (half-life 160 minutes). After intravenous adminstration of adenylate kinase to humans, a part of the activity was recovered in the urine within minutes. The potential use of assaying adenylate kinase levels for early diagnosis of myocardial infarction is discussed. Using various skeletal muscle diseases as examples, the possible use of the very rapid elimination of adenylate kinase from the serum in monitoring the course of the acute illnesses is described. The competitive inhibitor diadenosine pentaphosphate (AP5A) has a much higher affinity for the adenylate kinases from erythrocytes, heart or skeletal muscle than for the isoenzymes from liver or kidney. Therefore, AP5A can be used for the differential determination of adenylate kinase isoenzymes in the blood plasma or the urine.

  6. Cell cycle re-entry mechanisms after DNA damage checkpoints: giving it some gas to shut off the breaks!

    PubMed

    van Vugt, Marcel A T M; Yaffe, Michael B

    2010-06-01

    In order to maintain genetic integrity, cells are equipped with cell cycle checkpoints that detect DNA damage, orchestrate repair, and if necessary, eliminate severely damaged cells by inducing apoptotic cell death. The mitotic machinery is now emerging as an important determinant of the cellular responses to DNA damage where it functions as both the downstream target and the upstream regulator of the G2/M checkpoint. Cell cycle kinases and the DNA damage checkpoint kinases appear to reciprocally control each other. Specifically, cell cycle kinases control the inactivation of DNA damage checkpoint signaling. Termination of a DNA damage response by mitotic kinases appears to be an evolutionary conserved mechanism that allows resumption of cell cycle progression. Here we review recent reports in which molecular mechanisms underlying checkpoint silencing at the G2/M transition are elucidated.

  7. Inhibition of H3K9 methyltransferase G9a induces autophagy and apoptosis in oral squamous cell carcinoma

    SciTech Connect

    Ren, Aishu; Qiu, Yu; Affiliated Hospital of Stomatology, Chongqing Medical University, Chongqing, 401147

    2015-03-27

    Objective: To explore whether inhibition of H3K9 Methyltransferase G9a could exert an antitumoral effect in oral squamous cell carcinoma (OSCC). Materials and methods: First we checked G9a expression in two OSCC cell lines Tca8113 and KB. Next we used a special G9a inhibitor BIX01294 (BIX) to explore the effect of inhibition of G9a on OSCC in vitro. Cell growth was tested by typlan blue staining, MTT assay and Brdu immunofluorescence staining. Cell autophagy was examined by monodansylcadaverine (MDC) staining, LC3-II immunofluorescence staining and LC3-II western blot assay. Cell apoptosis was checked by FITC Annexin-V and PI labeling, tunnel staining and caspasemore » 3 western blot assay. Finally, the effect of inhibition of G9a on clonogenesis and tumorigenesis capacity of OSCC was analyzed by soft agar growth and xenograft model. Results: Here we showed that G9a was expressed in both Tca8113 and KB cells. Inhibition of G9a using BIX significantly reduced cell growth and proliferation in Tca8113 and KB. Inhibition of G9a induced cell autophagy with conversion of LC3-I to LC3-II and cell apoptosis with the expression of cleaved caspase 3. We also found that inhibition of G9a reduced colony formation in soft agar and repressed tumor growth in mouse xenograph model. Conclusion: Our results suggested that G9a might be a potential epigenetic target for OSCC treatment. - Highlights: • Inhibition of G9a reduced cell growth and proliferation in OSCC cells. • Inhibition of G9a induces autophagy and apoptosis in OSCC cells. • Inhibition of G9a repressed tumor growth in mouse xenograph model.« less

  8. Leucine reduces the proliferation of MC3T3-E1 cells through DNA damage and cell senescence.

    PubMed

    da Luz Dias, Raquel; Basso, Bruno; Donadio, Márcio Vinícius Fagundes; Pujol, Francesc Ventura; Bartrons, Ramón; Haute, Gabriela Viegas; Gassen, Rodrigo Benedetti; Bregolin, Henrique Dias; Krause, Gabriele; Viau, Cassiana; Saffi, Jenifer; Nunes, Fernanda Bordignon; Rosa, José Luis; de Oliveira, Jarbas Rodrigues

    2018-04-01

    Leucine (Leu) is an essential branched-chain amino acid, present in dairy products, which has been investigated for its important role in cell signaling. The effects of Leu on several kinds of cells have been studied, altough little is known on its action upon bone cells and cell proliferation. Thus, the aim of this study is to investigate the effects of Leu supplementation on the proliferation of pre-osteoblasts from MC3T3-E1 lineage. MC3T3-E1 cells were kept in Alpha medium supplemented with 10% fetal bovine serum and 1% antibiotic-antimitotic. Cells were treated during 48h by adding 50μM of Leu, which corresponds to a 12.5% increase of the amino acid in the culture medium. The evaluation of viability and proliferation of cultured cells was performed using Trypan Blue dye. In order to identify the mechanisms related to the decreased cellular proliferation, assays were performed to assess cytotoxicity, apotosis, oxidative stress, inflammation, autophagy, senescence and DNA damage. Results showed that Leu supplementation decreased cell proliferation by 40% through mechanisms not related to cell necrosis, apoptosis, oxidative stress, autophagy or inhibition of the mTORC1 pathway. On the other hand, Leu supplementation caused DNA damage. In conclusion, Leu caused a negative impact on bone cell proliferation by inducing cell senescence through DNA damage. Copyright © 2018 Elsevier Ltd. All rights reserved.

  9. Induction of cell cycle arrest, DNA damage, and apoptosis by nimbolide in human renal cell carcinoma cells.

    PubMed

    Hsieh, Yi-Hsien; Lee, Chien-Hsing; Chen, Hsiao-Yun; Hsieh, Shu-Ching; Lin, Chia-Liang; Tsai, Jen-Pi

    2015-09-01

    Nimbolide is a tetranortriterpenoid isolated from the leaves and flowers of Azadirachta indica which has been shown to exhibit anticancer, antioxidant, anti-inflammatory, and anti-invasive properties in a variety of cancer cells. However, the anti-tumor effect on human renal cell carcinoma (RCC) cells is unknown. In this study, we found that nimbolide treatment had a cytotoxic effect on 786-O and A-498 RCC cells in a dose-dependent manner. According to flow cytometric analysis, nimbolide treatment resulted in G2/M arrest in 786-O and A-498 cells accompanied with an increase in the phosphorylation status of p53, cdc2, cdc25c, and decreased expressions of cyclin A, cyclin B, cdc2, and cdc25c. Nimbolide also caused DNA damage in a dose-dependent manner as determined by comet assay and measurement of γ-H2AX. In addition, apoptotic cells were observed in an Annexin V-FITC/propidium iodide double-stained assay. The activities of caspase-3, -9, and poly ADP-ribose polymerase (PARP) were increased, and the expression of pro-caspase-8 was decreased in nimbolide-treated 786-O and A-498 cells. Western blot analysis revealed that the levels of intrinsic-related apoptotic proteins Bax and extrinsic-related proteins (DR5, CHOP) were significantly increased in nimbolide-treated 786-O and A-498 cells. In addition, the expressions of Bcl-2 and Mcl-1 were decreased in 786-O and A-498 cells after nimbolide treatment. We conclude that nimbolide can inhibit the growth of human RCC cells by inducing G2/M phase arrest by modulating cell cycle-related proteins and cell apoptosis by regulating intrinsic and extrinsic caspase signaling pathways. Nimbolide may be a promising therapeutic strategy for the treatment of RCC.

  10. Mitochondrial quality control in alveolar epithelial cells damaged byS. aureuspneumonia in mice.

    PubMed

    Suliman, Hagir B; Kraft, Bryan; Bartz, Raquel; Chen, Lingye; Welty-Wolf, Karen E; Piantadosi, Claude A

    2017-10-01

    Mitochondrial damage is often overlooked in acute lung injury (ALI), yet most of the lung's physiological processes, such as airway tone, mucociliary clearance, ventilation-perfusion (Va/Q) matching, and immune surveillance require aerobic energy provision. Because the cell's mitochondrial quality control (QC) process regulates the elimination and replacement of damaged mitochondria to maintain cell survival, we serially evaluated mitochondrial biogenesis and mitophagy in the alveolar regions of mice in a validated Staphylococcus aureus pneumonia model. We report that apart from cell lysis by direct contact with microbes, modest epithelial cell death was detected despite significant mitochondrial damage. Cell death by TdT-mediated dUTP nick-end labeling staining occurred on days 1 and 2 postinoculation: apoptosis shown by caspase-3 cleavage was present on days 1 and 2, while necroptosis shown by increased levels of phospho- mixed lineage kinase domain-like protein (MLKL) and receptor-interacting serine/threonine-protein kinase 1 (RIPK1) was present on day 1 Cell death in alveolar type I (AT 1 ) cells assessed by bronchoalveolar lavage fluid receptor for advanced glycation end points (RAGE) levels was high, yet AT 2 cell death was limited while both mitochondrial biogenesis and mitophagy were induced. These mitochondrial QC mechanisms were evaluated mainly in AT 2 cells by localizing increases in citrate synthase content, increases in nuclear mitochondrial biogenesis regulators nuclear respiratory factor-1 (NRF-1) and peroxisome proliferator-activated receptor-γ coactivator-1α (PGC-1α), and increases in light chain 3B protein (LC3-I)/LC3II ratios. Concomitant changes in p62, Pink 1, and Parkin protein levels indicated activation of mitophagy. By confocal microscopy, mitochondrial biogenesis and mitophagy were often observed on day 1 within the same AT 2 cells. These findings imply that mitochondrial QC activation in pneumonia-damaged AT 2 cells promotes cell

  11. Proton irradiation of stem cells: Radiation damage and chemical radioprotection

    NASA Technical Reports Server (NTRS)

    Riley, R. C.; Montour, J. L.; Gurney, C. W.

    1972-01-01

    Effects of high energy protons on erythropoietic stem cells and radioprotection by chemicals were investigated in NASA Space Radiation Effects Laboratory. The effects of a parallel beam of 600 MeV protons. The fluence, when converted to dose, were referenced to the synchrocyclotron beam monitors which were then used to administer radiation exposures. Mice were given graded doses to 300 rads to determine dose-response curve. Other mice received saline, AET, or 5-hydroxytryptamine 10 to 15 minutes before exposure.

  12. Ultraviolet Radiation-Induced Skin Aging: The Role of DNA Damage and Oxidative Stress in Epidermal Stem Cell Damage Mediated Skin Aging

    PubMed Central

    Panich, Uraiwan; Sittithumcharee, Gunya; Rathviboon, Natwarath

    2016-01-01

    Skin is the largest human organ. Skin continually reconstructs itself to ensure its viability, integrity, and ability to provide protection for the body. Some areas of skin are continuously exposed to a variety of environmental stressors that can inflict direct and indirect damage to skin cell DNA. Skin homeostasis is maintained by mesenchymal stem cells in inner layer dermis and epidermal stem cells (ESCs) in the outer layer epidermis. Reduction of skin stem cell number and function has been linked to impaired skin homeostasis (e.g., skin premature aging and skin cancers). Skin stem cells, with self-renewal capability and multipotency, are frequently affected by environment. Ultraviolet radiation (UVR), a major cause of stem cell DNA damage, can contribute to depletion of stem cells (ESCs and mesenchymal stem cells) and damage of stem cell niche, eventually leading to photoinduced skin aging. In this review, we discuss the role of UV-induced DNA damage and oxidative stress in the skin stem cell aging in order to gain insights into the pathogenesis and develop a way to reduce photoaging of skin cells. PMID:27148370

  13. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells

    PubMed Central

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N.; Guo, Lei; Mei, Nan

    2015-01-01

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition. PMID:26419945

  14. Etching and Damage Action on Microbes' Cells by Low Energy N+ Beam

    NASA Astrophysics Data System (ADS)

    Song, Dao-jun; Yu, Zeng-liang

    2000-08-01

    The action of etching and damage by 20 keV N+ beam on the cells of Deinococcus radiodurans and Escherichia coli was investigated by scanning electron microscope (SEM) and the electron spin resonance (ESR) spectrum of free radicals. The results showed that N+ implantation exerted the direct action of etching and damage of momentum transferring and the indirect action of the free radicals of energy deposition on their cells, many microholes were found on the surface of cells' wall and/or membrane by SEM, the damaged DNA was determined using DNA unwinding technique, and the signal of free radicals was measured by ESR. The degree of damage to cells by ion beam gradually increased with the increase implantation dose. With the post-treatment of 2 mmol/l caffeine and 0.5 mmol/l Na2-EDTA, the survival rate of D.radiodurans and E.coli further decreased in the order of caffeine > Na2-EDTA > control, and this suggested that low energy ion beam could be implanted into nucleus, doing a damage to DNA and resulting in the mutation of organisms.

  15. Ginkgo biloba leaf extract induces DNA damage by inhibiting topoisomerase II activity in human hepatic cells.

    PubMed

    Zhang, Zhuhong; Chen, Si; Mei, Hu; Xuan, Jiekun; Guo, Xiaoqing; Couch, Letha; Dobrovolsky, Vasily N; Guo, Lei; Mei, Nan

    2015-09-30

    Ginkgo biloba leaf extract has been shown to increase the incidence in liver tumors in mice in a 2-year bioassay conducted by the National Toxicology Program. In this study, the DNA damaging effects of Ginkgo biloba leaf extract and many of its constituents were evaluated in human hepatic HepG2 cells and the underlying mechanism was determined. A molecular docking study revealed that quercetin, a flavonoid constituent of Ginkgo biloba, showed a higher potential to interact with topoisomerase II (Topo II) than did the other Ginkgo biloba constituents; this in silico prediction was confirmed by using a biochemical assay to study Topo II enzyme inhibition. Moreover, as measured by the Comet assay and the induction of γ-H2A.X, quercetin, followed by keampferol and isorhamnetin, appeared to be the most potent DNA damage inducer in HepG2 cells. In Topo II knockdown cells, DNA damage triggered by Ginkgo biloba leaf extract or quercetin was dramatically decreased, indicating that DNA damage is directly associated with Topo II. DNA damage was also observed when cells were treated with commercially available Ginkgo biloba extract product. Our findings suggest that Ginkgo biloba leaf extract- and quercetin-induced in vitro genotoxicity may be the result of Topo II inhibition.

  16. Combusted but not smokeless tobacco products cause DNA damage in oral cavity cells.

    PubMed

    Gao, Hong; Prasad, G L; Zacharias, Wolfgang

    2014-05-01

    The aim of this work was to investigate genomic DNA damage in human oral cavity cells after exposure to different tobacco product preparations (TPPs). The oral carcinoma cell line 101A, gingival epithelial cells HGEC, and gingival fibroblasts HGF were exposed to TPM (total particulate matter from 3R4F cigarettes), ST/CAS (2S3 smokeless tobacco extract in complete artificial saliva), and NIC (nicotine). Treatments were for 24 h using TPM at its EC-50 doses, ST/CAS and NIC at doses with equi-nicotine units, and high doses for ST/CAS and NIC. Comet assays showed that TPM, but not ST/CAS or NIC, caused substantial DNA breaks in cells; only the high ST/CAS dose caused weak DNA damage. These results were confirmed by immunofluorescence for γ-H2AX protein. These data revealed that the combusted TPP caused substantial DNA damage in all cell types, whereas the two non-combusted TPPs exerted no or only minimal DNA damage. They support epidemiologic evidence on the relative risk associated with consumption of non-combusted versus combusted tobacco products, and help to understand potential genotoxic effects of such products on oral cavity cells. Copyright © 2014 Elsevier B.V. All rights reserved.

  17. Testosterone promotes DNA damage response under oxidative stress in prostate cancer cell lines.

    PubMed

    Ide, Hisamitsu; Lu, Yan; Yu, Jingsong; China, Toshiyuki; Kumamoto, Tomoka; Koseki, Tatsuro; Yamaguchi, Raizo; Muto, Satoru; Horie, Shigeo

    2012-09-15

    Sustained chronic inflammation and oxidative stress in the prostate promote prostate carcinogenesis. The process of oncogenic transformation leads to enhanced DNA damage and activates the checkpoint network that functions as an inducible barrier against cancer progression. Here, we analyzed the effects of testosterone on the DNA damage response in prostate cancer cells to assess whether testosterone functions a barrier to cancer progression under the oxidative stress. We examined the effects of testosterone on components of the DNA damage response pathway, including ATM (ataxia-telangiectasia-mutated kinase), H2AX (histone H2AX variant), and Chk2 (checkpoint kinase2) in prostate cancer cell lines, treated with various concentration of hydrogen peroxide (H(2) O(2) ). Cellular apoptosis was quantified by poly (ADP-ribose) polymerase (PARP) cleavage and flow cytometry. H(2) O(2) induced apoptosis and phosphorylation of ATM, Chk2, and H2AX in LNCaP cells. An ATM inhibitor, Ku55933, reduced H(2) O(2) -induced apoptosis in LNCaP and 22Rv1 cells. Androgen treatments increased H(2) O(2) -induced activation of the DNA damage response and PARP cleavage, but not when the H(2) O(2) -treated cells were also treated with the anti-androgen flutamide. The ATM inhibitor Ku55933 inhibited androgen-induced phosphorylation of ATM and PARP cleavage. DNA damage responses play important roles in the maintenance of the cell homeostasis in response to oxidative stress. Our results indicated that under oxidative stress androgen signaling may induce apoptosis by activating the DNA damage response. Copyright © 2012 Wiley Periodicals, Inc.

  18. Inhibition of HAS2 induction enhances the radiosensitivity of cancer cells via persistent DNA damage

    SciTech Connect

    Shen, Yan Nan; Shin, Hyun-Jin; Joo, Hyun-Yoo

    2014-01-17

    Highlights: •HAS2 may be a promising target for the radiosensitization of human cancer. •HAS2 is elevated (up to ∼10-fold) in irradiated radioresistant and -sensitive cancer cells. •HAS2 knockdown sensitizes cancer cells to radiation. •HAS2 knockdown potentiates irradiation-induced DNA damage and apoptotic death. •Thus, the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. -- Abstract: Hyaluronan synthase 2 (HAS2), a synthetic enzyme for hyaluronan, regulates various aspects of cancer progression, including migration, invasion and angiogenesis. However, the possible association of HAS2 with the response of cancer cells to anticancer radiotherapy, has not yet been elucidated. Here, we showmore » that HAS2 knockdown potentiates irradiation-induced DNA damage and apoptosis in cancer cells. Upon exposure to radiation, all of the tested human cancer cell lines exhibited marked (up to 10-fold) up-regulation of HAS2 within 24 h. Inhibition of HAS2 induction significantly reduced the survival of irradiated radioresistant and -sensitive cells. Interestingly, HAS2 depletion rendered the cells to sustain irradiation-induced DNA damage, thereby leading to an increase of apoptotic death. These findings indicate that HAS2 knockdown sensitizes cancer cells to radiation via persistent DNA damage, further suggesting that the irradiation-induced up-regulation of HAS2 contributes to the radioresistance of cancer cells. Thus, HAS2 could potentially be targeted for therapeutic interventions aimed at radiosensitizing cancer cells.« less

  19. Carcinoma cells misuse the host tissue damage response to invade the brain.

    PubMed

    Chuang, Han-Ning; van Rossum, Denise; Sieger, Dirk; Siam, Laila; Klemm, Florian; Bleckmann, Annalen; Bayerlová, Michaela; Farhat, Katja; Scheffel, Jörg; Schulz, Matthias; Dehghani, Faramarz; Stadelmann, Christine; Hanisch, Uwe-Karsten; Binder, Claudia; Pukrop, Tobias

    2013-08-01

    The metastatic colonization of the brain by carcinoma cells is still barely understood, in particular when considering interactions with the host tissue. The colonization comes with a substantial destruction of the surrounding host tissue. This leads to activation of damage responses by resident innate immune cells to protect, repair, and organize the wound healing, but may distract from tumoricidal actions. We recently demonstrated that microglia, innate immune cells of the CNS, assist carcinoma cell invasion. Here we report that this is a fatal side effect of a physiological damage response of the brain tissue. In a brain slice coculture model, contact with both benign and malignant epithelial cells induced a response by microglia and astrocytes comparable to that seen at the interface of human cerebral metastases. While the glial damage response intended to protect the brain from intrusion of benign epithelial cells by inducing apoptosis, it proved ineffective against various malignant cell types. They did not undergo apoptosis and actually exploited the local tissue reaction to invade instead. Gene expression and functional analyses revealed that the C-X-C chemokine receptor type 4 (CXCR4) and WNT signaling were involved in this process. Furthermore, CXCR4-regulated microglia were recruited to sites of brain injury in a zebrafish model and CXCR4 was expressed in human stroke patients, suggesting a conserved role in damage responses to various types of brain injuries. Together, our findings point to a detrimental misuse of the glial damage response program by carcinoma cells resistant to glia-induced apoptosis. Copyright © 2013 Wiley Periodicals, Inc.

  20. DNA Damage in Euonymus japonicus Leaf Cells Caused by Roadside Pollution in Beijing

    PubMed Central

    Li, Tianxin; Zhang, Minjie; Gu, Ke; Herman, Uwizeyimana; Crittenden, John; Lu, Zhongming

    2016-01-01

    The inhalable particles from vehicle exhaust can cause DNA damage to exposed organisms. Research on DNA damage is primarily focused on the influence of specific pollutants on certain species or the effect of environmental pollution on human beings. To date, little research has quantitatively studied the relationship between roadside pollution and DNA damage. Based on an investigation of the roadside pollution in Beijing, Euonymus japonicus leaves of differing ages grown in heavily-polluted sections were chosen as biomonitors to detect DNA damage using the comet assay technique. The percentage of DNA in the tail and tail moment was chosen as the analysis index based on SPSS data analysis. The roadside samples showed significantly higher levels of DNA damage than non-roadside samples, which increased in older leaves, and the DNA damage to Euonymus japonicus leaf cells was positively correlated with haze-aggravated roadside pollution. The correlation between damage and the Air Quality Index (AQI) are 0.921 (one-year-old leaves), 0.894 (two-year-old leaves), and 0.878 (three-year-old leaves). Over time, the connection between DNA damage and AQI weakened, with the sensitivity coefficient for δyear 1 being larger than δyear 2 and δyear 3. These findings support the suitability and sensitivity of the comet assay for surveying plants for an estimation of DNA damage induced by environmental genotoxic agents. This study might be applied as a preliminary quantitative method for Chinese urban air pollution damage assessment caused by environmental stress. PMID:27455298

  1. Generation of Reactive Oxygen Species by Polyenylpyrroles Derivatives Causes DNA Damage Leading to G2/M Arrest and Apoptosis in Human Oral Squamous Cell Carcinoma Cells

    PubMed Central

    Hua, Kuo-Feng; Liao, Pei-Chun; Fang, Zhanxiong; Yang, Feng-Ling; Yang, Yu-Liang; Chen, Yi-Lin; Chiu, Yi-Chich; Liu, May-Lan; Lam, Yulin; Wu, Shih-Hsiung

    2013-01-01

    Oral squamous cell carcinoma (OSCC) accounts for 5.8% of all malignancies in Taiwan and the incidence of OSCC is on the rise. OSCC is also a common malignancy worldwide and the five-year survival rate remains poor. Therefore, new and effective treatments are needed to control OSCC. In the present study we have investigated the efficacy and associated mechanisms of polyenylpyrroles and their analogs in both in vitro cell culture and in vivo nude mice xenografts. Auxarconjugatin B (compound 1a) resulted in cell cycle arrest in the G2/M phase and caspase-dependent apoptosis in OEC-M1 and HSC-3 cells by activating DNA damage and mitochondria dysfunction through the loss of mitochondrial membrane potential, release of cytochrome c, increase in B-cell lymphoma-2-associated X protein level, and decrease in B-cell lymphoma-2 level. Compound 1a-induced generation of intracellular reactive oxygen species through cytochrome P450 1A1 was identified as a major mechanism of its effect for DNA damage, mitochondria dysfunction and apoptosis, which was reversed by antioxidant N-acetylcysteine as well as cytochrome P450 1A1 inhibitor and specific siRNA. Furthermore, compound 1a-treated nude mice showed a reduction in the OEC-M1 xenograft tumor growth and an increase in the caspase-3 activation in xenograft tissue. These results provide promising insights as to how compound 1a mediates cytotoxicity and may prove to be a molecular rationale for its translation into a potential therapeutic against OSCC. PMID:23840748

  2. Phospho-Ser/Thr-binding domains: navigating the cell cycle and DNA damage response.

    PubMed

    Reinhardt, H Christian; Yaffe, Michael B

    2013-09-01

    Coordinated progression through the cell cycle is a complex challenge for eukaryotic cells. Following genotoxic stress, diverse molecular signals must be integrated to establish checkpoints specific for each cell cycle stage, allowing time for various types of DNA repair. Phospho-Ser/Thr-binding domains have emerged as crucial regulators of cell cycle progression and DNA damage signalling. Such domains include 14-3-3 proteins, WW domains, Polo-box domains (in PLK1), WD40 repeats (including those in the E3 ligase SCF(βTrCP)), BRCT domains (including those in BRCA1) and FHA domains (such as in CHK2 and MDC1). Progress has been made in our understanding of the motif (or motifs) that these phospho-Ser/Thr-binding domains connect with on their targets and how these interactions influence the cell cycle and DNA damage response.

  3. Fibroblasts Cultured on Nanowires Exhibit Low Motility, Impaired Cell Division, and DNA Damage

    PubMed Central

    Persson, Henrik; Købler, Carsten; Mølhave, Kristian; Samuelson, Lars; Tegenfeldt, Jonas O; Oredsson, Stina; Prinz, Christelle N

    2013-01-01

    Nanowires are commonly used as tools for interfacing living cells, acting as biomolecule-delivery vectors or electrodes. It is generally assumed that the small size of the nanowires ensures a minimal cellular perturbation, yet the effects of nanowires on cell migration and proliferation remain largely unknown. Fibroblast behaviour on vertical nanowire arrays is investigated, and it is shown that cell motility and proliferation rate are reduced on nanowires. Fibroblasts cultured on long nanowires exhibit failed cell division, DNA damage, increased ROS content and respiration. Using focused ion beam milling and scanning electron microscopy, highly curved but intact nuclear membranes are observed, showing no direct contact between the nanowires and the DNA. The nanowires possibly induce cellular stress and high respiration rates, which trigger the formation of ROS, which in turn results in DNA damage. These results are important guidelines to the design and interpretation of experiments involving nanowire-based transfection and electrical characterization of living cells. PMID:23813871

  4. A simple model of space radiation damage in GaAs solar cells

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; Stith, J. J.; Stock, L. V.

    1983-01-01

    A simple model is derived for the radiation damage of shallow junction gallium arsenide (GaAs) solar cells. Reasonable agreement is found between the model and specific experimental studies of radiation effects with electron and proton beams. In particular, the extreme sensitivity of the cell to protons stopping near the cell junction is predicted by the model. The equivalent fluence concept is of questionable validity for monoenergetic proton beams. Angular factors are quite important in establishing the cell sensitivity to incident particle types and energies. A fluence of isotropic incidence 1 MeV electrons (assuming infinite backing) is equivalent to four times the fluence of normal incidence 1 MeV electrons. Spectral factors common to the space radiations are considered, and cover glass thickness required to minimize the initial damage for a typical cell configuration is calculated. Rough equivalence between the geosynchronous environment and an equivalent 1 MeV electron fluence (normal incidence) is established.

  5. Establishment of a blue light damage model of human retinal pigment epithelial cells in vitro.

    PubMed

    Su, G; Cai, S J; Gong, X; Wang, L L; Li, H H; Wang, L M

    2016-06-24

    To establish a blue-light damage model of human retinal pigment epithelium (RPE). Fourth-generation human RPE cells were randomly divided into two groups. In group A, cells were exposed to blue light (2000 ± 500 lux) for 0 (control), 3, 6, 9, and 12 h, and cell culture was stopped after 12 h. In group B, cells were exposed to blue light at the same intensity and time periods, but cell culture was stopped after 24 h. TdT-mediated dUTP nick-end labeling (TUNEL) assay was performed to determine the most suitable illuminating time with apoptotic index. Flow cytometry was used to determine apoptotic ratio of RPEs. In group A, the apoptotic index of cells that received 6, 9 and 12 h of blue light was higher than that of control. The apoptotic index of cells receiving 9 and 12 h was higher than that of 6 h (P = 0.000). In group B, the apoptotic index and RPE cell apoptosis ratio of cells exposed to 6, 9 and 12 h of blue light were higher than that of 3 h (P = 0.000); and cells receiving 9 and 12 h had higher values than that of 6 h. This study demonstrated that the best conditions to establish a blue light damage model of human retinal pigment epithelial cells in vitro are 2000 ± 500 lux light intensity for 6 h, with 24 h of cell culture post-exposure.

  6. A concise review of DNA damage checkpoints and repair in mammalian cells.

    PubMed

    Houtgraaf, Jaco H; Versmissen, Jorie; van der Giessen, Wim J

    2006-01-01

    DNA of eukaryotic cells, including vascular cells, is under the constant attack of chemicals, free radicals, or ionizing radiation that can be caused by environmental exposure, by-products of intracellular metabolism, or medical therapy. Damage may be either limited to altered DNA bases and abasic sites or extensive like double-strand breaks (DSBs). Nuclear proteins sense this damage and initiate the attachment of protein complexes at the site of the lesion. Subsequently, signal transducers, mediators, and finally, effector proteins phosphorylate targets (e.g., p53) that eventually results in cell cycle arrest at the G1/S, intra-S, or G2/M checkpoint until the lesion undergoes repair. Defective cell cycle arrest at the respective checkpoints is associated with genome instability and oncogenesis. When cell cycle arrest is accomplished, the DNA repair machinery can become effective. Important pathways in mammalian cells are the following: base excision repair, nucleotide excision repair, mismatch repair, and DSB repair. When repair is successful, the cell cycle arrest may be lifted. If repair is unsuccessful (e.g., by high doses of DNA-damaging agents or genetic defects in the DNA repair machinery), then this may lead to permanent cell cycle arrest (cellular senescence), apoptosis, or oncogenesis.

  7. Extracellular vesicles derived from human vestibular schwannomas associated with poor hearing damage cochlear cells.

    PubMed

    Soares, Vitor Y R; Atai, Nadia A; Fujita, Takeshi; Dilwali, Sonam; Sivaraman, Sarada; Landegger, Lukas D; Hochberg, Fred H; Oliveira, Carlos A P C; Bahmad, Fayez; Breakefield, Xandra O; Stankovic, Konstantina M

    2016-11-01

    Vestibular schwannoma (VS) is a tumor of the vestibular nerve that transmits balance information from the inner ear to the brain. Sensorineural hearing loss occurs in 95% of patients with these tumors, but the cause of this loss is not well understood. We posit a role of VS-secreted extracellular vesicles (EVs) as a major contributing factor in cochlear nerve damage. Using differential centrifugation, we isolated EVs from VS cell line HEI-193 and primary cultured human VS cells from patients with good hearing or poor hearing. The EVs were characterized using a Nanosight device and transmission electron microscopy and by extracting their RNA content. The EVs' effects on cultured murine spiral ganglion cells and organotypic cochlear cultures were studied using a transwell dual-culture system and by direct labeling of EVs with PKH-67 dye. EV-induced changes in cochlear cells were quantified using confocal immunohistochemistry. Transfection of VS cells with a green fluorescent protein-containing plasmid was confirmed with reverse transcription PCR. Human VS cells, from patients with poor hearing, produced EVs that could damage both cultured murine cochlear sensory cells and neurons. In contrast, EVs derived from VS cells from patients with good hearing did not damage the cultured cochlear cells. This is the first report on EVs derived from VSs and on the capacity of EVs from VSs from patients with hearing loss to selectively damage cochlear cells, thereby identifying a potential novel mechanism of VS-associated sensorineural hearing loss. © The Author(s) 2016. Published by Oxford University Press on behalf of the Society for Neuro-Oncology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  8. Correlation of electron and proton irradiation-induced damage in InP solar cells

    NASA Technical Reports Server (NTRS)

    Walters, Robert J.; Summers, Geoffrey P.; Messenger, Scott R.; Burke, Edward A.

    1995-01-01

    When determining the best solar cell technology for a particular space flight mission, accurate prediction of solar cell performance in a space radiation environment is essential. The current methodology used to make such predictions requires extensive experimental data measured under both electron and proton irradiation. Due to the rising cost of accelerators and irradiation facilities, such extensive data sets are expensive to obtain. Moreover, with the rapid development of novel cell designs, the necessary data are often not available. Therefore, a method for predicting cell degradation based on limited data is needed. Such a method has been developed at the Naval Research Laboratory based on damage correlation using 'displacement damage dose' which is the product of the non-ionizing energy loss (NIEL) and the particle fluence. Displacement damage dose is a direct analog of the ionization dose used to correlate the effects of ionizing radiations. In this method, the performance of a solar cell in a complex radiation environment can be predicted from data on a single proton energy and two electron energies, or one proton energy, one electron energy, and Co(exp 60) gammas. This method has been used to accurately predict the extensive data set measured by Anspaugh on GaAs/Ge solar cells under a wide range of electron and proton energies. In this paper, the method is applied to InP solar cells using data measured under 1 MeV electron and 3 MeV proton irradiations, and the calculations are shown to agree well with the measured data. In addition to providing accurate damage predictions, this method also provides a basis for quantitative comparisons of the performance of different cell technologies. The performance of the present InP cells is compared to that published for GaAs/Ge cells. The results show InP to be inherently more resistant to displacement energy deposition than GaAs/Ge.

  9. Carboxylated nanodiamonds inhibit γ-irradiation damage of human red blood cells

    NASA Astrophysics Data System (ADS)

    Santacruz-Gomez, K.; Silva-Campa, E.; Melendrez-Amavizca, R.; Teran Arce, F.; Mata-Haro, V.; Landon, P. B.; Zhang, C.; Pedroza-Montero, M.; Lal, R.

    2016-03-01

    Nanodiamonds when carboxylated (cNDs) act as reducing agents and hence could limit oxidative damage in biological systems. Gamma (γ)-irradiation of whole blood or its components is required in immunocompetent patients to prevent transfusion-associated graft versus host disease (TA-GVHD). However, γ-irradiation of blood also deoxygenates red blood cells (RBCs) and induces oxidative damage, including abnormalities in cellular membranes and hemolysis. Using atomic force microscopy (AFM) and Raman spectroscopy, we examined the effect of cNDs on γ-irradiation mediated deoxygenation and morphological damage of RBCs. γ-Radiation induced several morphological phenotypes, including stomatocytes, codocytes and echinocytes. While stomatocytes and codocytes are reversibly damaged RBCs, echinocytes are irreversibly damaged. AFM images show significantly fewer echinocytes among cND-treated γ-irradiated RBCs. The Raman spectra of γ-irradiated RBCs had more oxygenated hemoglobin patterns when cND-treated, resembling those of normal, non-irradiated RBCs, compared to the non-cND-treated RBCs. cND inhibited hemoglobin deoxygenation and morphological damage, possibly by neutralizing the free radicals generated during γ-irradiation. Thus cNDs have the therapeutic potential to preserve the quality of stored blood following γ-irradiation.Nanodiamonds when carboxylated (cNDs) act as reducing agents and hence could limit oxidative damage in biological systems. Gamma (γ)-irradiation of whole blood or its components is required in immunocompetent patients to prevent transfusion-associated graft versus host disease (TA-GVHD). However, γ-irradiation of blood also deoxygenates red blood cells (RBCs) and induces oxidative damage, including abnormalities in cellular membranes and hemolysis. Using atomic force microscopy (AFM) and Raman spectroscopy, we examined the effect of cNDs on γ-irradiation mediated deoxygenation and morphological damage of RBCs. γ-Radiation induced several

  10. Soft X-Ray Microscopy Radiation Damage On Fixed Cells Investigated With Synchrotron Radiation FTIR Microscopy.

    PubMed

    Gianoncelli, A; Vaccari, L; Kourousias, G; Cassese, D; Bedolla, D E; Kenig, S; Storici, P; Lazzarino, M; Kiskinova, M

    2015-05-14

    Radiation damage of biological samples remains a limiting factor in high resolution X-ray microscopy (XRM). Several studies have attempted to evaluate the extent and the effects of radiation damage, proposing strategies to minimise or prevent it. The present work aims to assess the impact of soft X-rays on formalin fixed cells on a systematic manner. The novelty of this approach resides on investigating the radiation damage not only with XRM, as often reported in relevant literature on the topic, but by coupling it with two additional independent non-destructive microscopy methods: Atomic Force Microscopy (AFM) and FTIR Microscopy (FTIRM). Human Embryonic Kidney 293 cells were exposed to different radiation doses at 1 keV. In order to reveal possible morphological and biochemical changes, the irradiated cells were systematically analysed with AFM and FTIRM before and after. Results reveal that while cell morphology is not substantially affected, cellular biochemical profile changes significantly and progressively when increasing dose, resulting in a severe breakdown of the covalent bonding network. This information impacts most soft XRM studies on fixed cells and adds an in-depth understanding of the radiation damage for developing better prevention strategies.

  11. Improved staining method for determining the extent of thermal damage to cells.

    PubMed

    Sherwood, Margaret E; Flotte, Thomas J

    2007-02-01

    Enzyme histochemical stains of frozen sections have been used by investigators to assess thermal damage. The assessment of thermal damage to cells in lipid-rich tissues such as subcutaneous tissue and sebaceous glands can be difficult due to the quality of frozen sections of such tissues. The purpose of this study is to develop an improved method for this type of evaluation. Thick frozen sections of thermally damaged pig and human skin were stained for lactate dehydrogenase. The sections were fixed in formalin and processed for paraffin-embedded sections. The sections showed well-defined localization of the enzymatic deposits as well as preservation of the tissue architecture. The paraffin-embedded lactate dehydrogenase stained sections provide improved evaluation of thermally damaged tissues, particularly the lipid rich tissues. (c) 2007 Wiley-Liss, Inc.

  12. Pyrroloquinoline quinone protects mouse brain endothelial cells from high glucose-induced damage in vitro

    PubMed Central

    Wang, Zhong; Chen, Guo-qiang; Yu, Gui-ping; Liu, Chang-jian

    2014-01-01

    Aim: To investigate the effects of pyrroloquinoline quinone (PQQ), an oxidoreductase cofactor, on high glucose-induced mouse endothelial cell damage in vitro. Methods: Mouse brain microvascular endothelial bEND.3 cells were exposed to different glucose concentrations (5.56, 25 and 40 mmol/L) for 24 or 48 h. The cell viability was examined using MTT assay. Flow cytometry was used to analyze the apoptosis and ROS levels in the cells. MitoTracker Green staining was used to examine the mitochondria numbers in the cells. Western blot analysis was used to analyze the expression of HIF-1α and the proteins in JNK pathway. Results: Treatment of bEND.3 cells with high glucose significantly decreased the cell viability, while addition of PQQ (1 and 10 μmol/L) reversed the high glucose-induced cell damage in a concentration-dependent manner. Furthermore, PQQ (100 μmol/L) significantly suppressed the high glucose-induced apoptosis and ROS production in the cells. PQQ significantly reversed the high glucose-induced reduction in both the mitochondrial membrane potential and mitochondria number in the cells. The high glucose treatment significantly increased the expression of HIF-1α and JNK phosphorylation in the cells, and addition of PQQ led to a further increase of HIF-1α level and a decrease of JNK phosphorylation. Addition of JNK inhibitor SP600125 (10 μmol/L) also significantly suppressed high glucose-induced apoptosis and JNK phosphorylation in bEND.3 cells. Conclusion: PQQ protects mouse brain endothelial cells from high glucose damage in vitro by suppressing intracellular ROS and apoptosis via inhibiting JNK signaling pathway. PMID:25283505

  13. DNA damage and the bystander response in tumor and normal cells exposed to X-rays.

    PubMed

    Subhashree, M; Venkateswarlu, R; Karthik, K; Shangamithra, V; Venkatachalam, P

    2017-09-01

    Monolayer and suspension cultures of tumor (BMG-1, CCRF-CEM), normal (AG1522, HADF, lymphocytes) and ATM-mutant (GM4405) human cells were exposed to X-rays at doses used in radiotherapy (high dose and high dose-rate) or radiological imaging (low dose and low dose-rate). Radiation-induced DNA damage, its persistence, and possible bystander effects were evaluated, based on DNA damage markers (γ-H2AX, p53 ser15 ) and cell-cycle-specific cyclins (cyclin B1 and cyclin D1). Dose-dependent DNA damage and a dose-independent bystander response were seen after exposure to high dose and high dose-rate radiation. The level of induced damage (expression of p53 ser15 , γ-H2AX) depended on ATM status. However, low dose and dose-rate exposures neither increased expression of marker proteins nor induced a bystander response, except in the CCRF-CEM cells. Bystander effects after high-dose irradiation may contribute to stochastic and deterministic effects. Precautions to protect unexposed regions or to inhibit transmission of DNA damage signaling might reduce radiation risks. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Oxidative DNA damage and repair in a cell lineage model of human proliferative breast disease (PBD).

    PubMed

    Starcevic, Susan L; Diotte, Nicole M; Zukowski, Kim L; Cameron, Mark J; Novak, Raymond F

    2003-09-01

    Oxidative damage to DNA is thought to play a significant role in mutagenesis, aging, and cancer. Sensitivity to oxidative DNA damage and DNA repair efficiency were examined using a series of human breast epithelial cell lines-MCF-10A, MCF-10AT, and MCF-10ATG3B-with progressively elevated Ras protein. Breast epithelial cells were treated with H2O2, in the absence and presence of the DNA-repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C). DNA strand breaks were assessed by the mean olive tail moment (microm) using the alkaline single-cell gel electrophoresis (Comet) assay. In untreated cells, the mean olive tail moment values were 4.3 +/- 0.7, 8.3 +/- 1.1, and 7.1 +/- 0.6 microm in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively. Five min H2O2 treatment produced concentration-dependent DNA damage, with the MCF-10A cells most susceptible and the tumorigenic MCF-10ATG3B cells the least susceptible. Treatment with 100 microM H2O2 resulted in approximately 17-, 6-, and 4.5-fold increases in mean olive tail moment values in the MCF-10A, MCF-10AT, and MCF-10ATG3B cells, respectively, compared to untreated cells. The HCC1937 tumor cell line responded in a manner comparable to the MCF-10ATG3B cells treated with H2O2, HU/Ara-C pre-treatment resulted in a approximately 1.5-fold increase in olive tail moment values in all three cell lines. Protein levels of antioxidant enzymes, including catalase, copper/zinc superoxide dismutase (Cu/Zn SOD), and manganese SOD (MnSOD) were determined in order to examine a potential mechanism for increased resistance to H2O2-mediated DNA damage. Levels of these enzymes increased progressively, with highest expression in MCF-10ATG3B cells. Increased cellular resistance also coincided with marked decreases in p53 protein levels. These results demonstrate that, in this cell lineage, sensitivity to oxidative DNA damage by H2O2 decreases with tumorigenicity (i.e., MCF-10A vs. MCF-10ATG3B), and show that DNA repair, altered

  15. DNA damage signaling regulates age-dependent proliferative capacity of quiescent inner ear supporting cells

    PubMed Central

    Laos, Maarja; Anttonen, Tommi; Kirjavainen, Anna; Hällström, Taija af; Laiho, Marikki; Pirvola, Ulla

    2014-01-01

    Supporting cells (SCs) of the cochlear (auditory) and vestibular (balance) organs hold promise as a platform for therapeutic regeneration of the sensory hair cells. Prior data have shown proliferative restrictions of adult SCs forced to re-enter the cell cycle. By comparing juvenile and adult SCs in explant cultures, we have here studied how proliferative restrictions are linked with DNA damage signaling. Cyclin D1 overexpression, used to stimulate cell cycle re-entry, triggered higher proliferative activity of juvenile SCs. Phosphorylated form of histone H2AX (γH2AX) and p53 binding protein 1 (53BP1) were induced in a foci-like pattern in SCs of both ages as an indication of DNA double-strand break formation and activated DNA damage response. Compared to juvenile SCs, γH2AX and the repair protein Rad51 were resolved with slower kinetics in adult SCs, accompanied by increased apoptosis. Consistent with the in vitro data, in a Rb mutant mouse model in vivo, cell cycle re-entry of SCs was associated with γH2AX foci induction. In contrast to cell cycle reactivation, pharmacological stimulation of SC-to-hair-cell transdifferentiation in vitro did not trigger γH2AX. Thus, DNA damage and its prolonged resolution are critical barriers in the efforts to stimulate proliferation of the adult inner ear SCs. PMID:25063730

  16. Potentiated DNA Damage Response in Circulating Breast Tumor Cells Confers Resistance to Chemotherapy*

    PubMed Central

    Gong, Chang; Liu, Bodu; Yao, Yandan; Qu, Shaohua; Luo, Wei; Tan, Weige; Liu, Qiang; Yao, Herui; Zou, Lee; Su, Fengxi; Song, Erwei

    2015-01-01

    Circulating tumor cells (CTCs) are seeds for cancer metastasis and are predictive of poor prognosis in breast cancer patients. Whether CTCs and primary tumor cells (PTCs) respond to chemotherapy differently is not known. Here, we show that CTCs of breast cancer are more resistant to chemotherapy than PTCs because of potentiated DNA repair. Surprisingly, the chemoresistance of CTCs was recapitulated in PTCs when they were detached from the extracellular matrix. Detachment of PTCs increased the levels of reactive oxygen species and partially activated the DNA damage checkpoint, converting PTCs to a CTC-like state. Inhibition of checkpoint kinases Chk1 and Chk2 in CTCs reduces the basal checkpoint response and sensitizes CTCs to DNA damage in vitro and in mouse xenografts. Our results suggest that DNA damage checkpoint inhibitors may benefit the chemotherapy of breast cancer patients by suppressing the chemoresistance of CTCs and reducing the risk of cancer metastasis. PMID:25897074

  17. Proposed Pharmacological Countermeasures Against Apoptotic Cell Death in Experimental Models Mimicking Space Environment Damage

    NASA Astrophysics Data System (ADS)

    Lulli, Matteo; Papucci, Laura; Witort, Ewa; Donnini, Martino; Lapucci, Andrea; Lazzarano, Stefano; Mazzoni, Tiziano; Simoncini, Madine; Falciani, Piergiuseppe; Capaccioli, Sergio

    2008-06-01

    Several damaging agents have been suggested to affect human vision during long term space travels. Recently, apoptosis induced by DNA-damaging agents has emerged as frequent pathogenetic mechanism of ophthalmologic pathologies. Here, we propose two countermeasures: coenzyme Q10 and bcl-2 downregulation preventing antisense oligoribonucleotides (ORNs), aimed to inhibit cellular apoptotic death. Our studies have been carried out on retina and neuronal cultured cells treated with the following apoptotic stimuli mimicking space environment: a several-day exposure to either 3H-labeled tymidine or to the genotoxic drug doxorubicin, UV irradiation, hypoxia and glucose/growth factor starvation (Locke medium). The preliminary results clearly indicate that CoQ10, as well as bcl-2 down-regulation preventing ORNs, significantly counteract apoptosis in response to different DNA damaging agents in cultured eye and in neuronal cells. This supports the possibility that both could be optimal countermeasures against ophthalmologic lesions during space explorations.

  18. Cell phenotypic variation in normal and damaged tendons

    PubMed Central

    Clegg, Peter D; Strassburg, Sandra; Smith, Roger K

    2007-01-01

    Injuries to tendons are common in both human athletes as well as in animals, such as the horse, which are used for competitive purposes. Furthermore, such injuries are also increasing in prevalence in the ageing, sedentary population. Tendon diseases often respond poorly to treatment and require lengthy periods of rehabilitation. The tendon has a unique extracellular matrix, which has developed to withstand the mechanical demands of such tensile-load bearing structures. Following injury, any repair process is inadequate and results in tissue that is distinct from original tendon tissue. There is growing evidence for the key role of the tendon cell (tenocyte) in both the normal physiological homeostasis and regulation of the tendon matrix and the pathological derangements that occur in disease. In particular, the tenocyte is considered to have a major role in effecting the subclinical matrix degeneration that is thought to occur prior to clinical disease, as well as in the severe degradative events that occur in the tendon at the onset of clinical disease. Furthermore, the tenocyte is likely to have a central role in the production of the biologically inadequate fibrocartilaginous repair tissue that develops subsequent to tendinopathy. Understanding the biology of the tenocyte is central to the development of appropriate interventions and drug therapies that will either prevent the onset of disease, or lead to more rapid and appropriate repair of injured tendon. Central to this is a full understanding of the proteolytic response in the tendon in disease by such enzymes as metalloproteinases, as well as the control of the inappropriate fibrocartilaginous differentiation. Finally, it is important that we understand the role of both intrinsic and extrinsic cellular elements in the repair process in the tendon subsequent to injury. PMID:17696903

  19. DNA Damage Response and Spindle Assembly Checkpoint Function throughout the Cell Cycle to Ensure Genomic Integrity

    PubMed Central

    Lawrence, Katherine S.; Chau, Thinh; Engebrecht, JoAnne

    2015-01-01

    Errors in replication or segregation lead to DNA damage, mutations, and aneuploidies. Consequently, cells monitor these events and delay progression through the cell cycle so repair precedes division. The DNA damage response (DDR), which monitors DNA integrity, and the spindle assembly checkpoint (SAC), which responds to defects in spindle attachment/tension during metaphase of mitosis and meiosis, are critical for preventing genome instability. Here we show that the DDR and SAC function together throughout the cell cycle to ensure genome integrity in C. elegans germ cells. Metaphase defects result in enrichment of SAC and DDR components to chromatin, and both SAC and DDR are required for metaphase delays. During persistent metaphase arrest following establishment of bi-oriented chromosomes, stability of the metaphase plate is compromised in the absence of DDR kinases ATR or CHK1 or SAC components, MAD1/MAD2, suggesting SAC functions in metaphase beyond its interactions with APC activator CDC20. In response to DNA damage, MAD2 and the histone variant CENPA become enriched at the nuclear periphery in a DDR-dependent manner. Further, depletion of either MAD1 or CENPA results in loss of peripherally associated damaged DNA. In contrast to a SAC-insensitive CDC20 mutant, germ cells deficient for SAC or CENPA cannot efficiently repair DNA damage, suggesting that SAC mediates DNA repair through CENPA interactions with the nuclear periphery. We also show that replication perturbations result in relocalization of MAD1/MAD2 in human cells, suggesting that the role of SAC in DNA repair is conserved. PMID:25898113

  20. Effects of sodium bicarbonate on butyric acid-induced epithelial cell damage in vitro.

    PubMed

    Takigawa, Satoko; Sugano, Naoyuki; Ochiai, Kuniyasu; Arai, Noriyuki; Ota, Noriko; Ito, Koichi

    2008-12-01

    Butyric acid is detected in periodontal pockets and is thought to be involved in the initiation and progression of periodontal disease. We examined the effects of sodium bicarbonate on the butyric acid-induced epithelial cell damage. The human gingival carcinoma cell line Ca9-22 was cultured in medium that contained butyric acid with or without sodium bicarbonate. The viability of cells treated with sodium bicarbonate was significantly higher than that of cells treated with butyric acid alone. The effects of butyric acid on ICAM-1 expression were significantly improved by sodium bicarbonate. Within the limitations of this in vitro study, sodium bicarbonate was indicated to be a useful therapeutic agent to reduce the butyric acid-induced periodontal tissue damage.

  1. Circulating blood cells function as a surveillance system for damaged tissue in Drosophila larvae

    PubMed Central

    Babcock, Daniel T.; Brock, Amanda R.; Fish, Greg S.; Wang, Yan; Perrin, Laurent; Krasnow, Mark A.; Galko, Michael J.

    2008-01-01

    Insects have an open circulatory system in which the heart pumps blood (hemolymph) into the body cavity, where it directly bathes the internal organs and epidermis. The blood contains free and tissue-bound immune cells that function in the inflammatory response. Here, we use live imaging of transgenic Drosophila larvae with fluorescently labeled blood cells (hemocytes) to investigate the circulatory dynamics of larval blood cells and their response to tissue injury. We find that, under normal conditions, the free cells rapidly circulate, whereas the tissue-bound cells are sessile. After epidermal wounding, tissue-bound cells around the wound site remain sessile and unresponsive, whereas circulating cells are rapidly recruited to the site of damage by adhesive capture. After capture, these cells distribute across the wound, appear phagocytically active, and are subsequently released back into circulation by the healing epidermis. The results demonstrate that circulating cells function as a surveillance system that monitors larval tissues for damage, and that adhesive capture, an important mechanism of recruitment of circulating cells to inflammatory sites in vertebrates, is shared by insects and vertebrates despite the vastly different architectures of their circulatory systems. PMID:18632567

  2. Protective role of quercetin against cisplatin-induced hair cell damage in zebrafish embryos.

    PubMed

    Lee, S K; Oh, K H; Chung, A Y; Park, H C; Lee, S H; Kwon, S Y; Choi, J

    2015-11-01

    The aim of this study was to evaluate the protective effects of quercetin on cisplatin-induced hair cell damage in transgenic zebrafish embryos. Five days postfertilization zebrafish embryos were exposed to 1 mM cisplatin and quercetin at 10, 50, 100, or 200 μM for 4 h. Hair cells within neuromasts of the supraorbital, otic, and occipital lateral lines were analyzed by fluorescent microscopy (n = 10). Survival of hair cells was calculated as the average number of hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy. Hair cell damage in neuromasts was decreased by co-treatment of quercetin and cisplatin (quercetin 100 μM: 8.6 ± 1.1 cells; 1 mM cisplatin only: 5.0 ± 0.5 cells; n = 10, p < 0.05); apoptosis of hair cells examined by special stain was also decreased by quercetin. The ultrastructure of hair cells within neuromasts was preserved in zebrafish by the combination of quercetin (100 μM) and cisplatin (1 mM). In conclusion, quercetin showed protective effects against cisplatin-induced toxicity in a zebrafish model. The results of this study suggest the possibility of a protective role of quercetin against cisplatin-induced apoptotic cell death in zebrafish. © The Author(s) 2015.

  3. Gene 33/Mig6 inhibits hexavalent chromium-induced DNA damage and cell transformation in human lung epithelial cells.

    PubMed

    Park, Soyoung; Li, Cen; Zhao, Hong; Darzynkiewicz, Zbigniew; Xu, Dazhong

    2016-02-23

    Hexavalent Chromium [Cr(VI)] compounds are human lung carcinogens and environmental/occupational hazards. The molecular mechanisms of Cr(VI) carcinogenesis appear to be complex and are poorly defined. In this study, we investigated the potential role of Gene 33 (ERRFI1, Mig6), a multifunctional adaptor protein, in Cr(VI)-mediated lung carcinogenesis. We show that the level of Gene 33 protein is suppressed by both acute and chronic Cr(VI) treatments in a dose- and time-dependent fashion in BEAS-2B lung epithelial cells. The inhibition also occurs in A549 lung bronchial carcinoma cells. Cr(VI) suppresses Gene 33 expression mainly through post-transcriptional mechanisms, although the mRNA level of gene 33 also tends to be lower upon Cr(VI) treatments. Cr(VI)-induced DNA damage appears primarily in the S phases of the cell cycle despite the high basal DNA damage signals at the G2M phase. Knockdown of Gene 33 with siRNA significantly elevates Cr(VI)-induced DNA damage in both BEAS-2B and A549 cells. Depletion of Gene 33 also promotes Cr(VI)-induced micronucleus (MN) formation and cell transformation in BEAS-2B cells. Our results reveal a novel function of Gene 33 in Cr(VI)-induced DNA damage and lung epithelial cell transformation. We propose that in addition to its role in the canonical EGFR signaling pathway and other signaling pathways, Gene 33 may also inhibit Cr(VI)-induced lung carcinogenesis by reducing DNA damage triggered by Cr(VI).

  4. Nickel Oxide Nanoparticles Induce Oxidative DNA Damage and Apoptosis in Kidney Cell Line (NRK-52E).

    PubMed

    Abudayyak, Mahmoud; Guzel, Elif; Özhan, Gül

    2017-07-01

    Increasing use of nickel oxide (NiO) nanoparticles in different applications results in high occupational and environmental exposure to them. However, the effect of NiO nanoparticles on human health is still poorly documented. It was aimed to investigate the toxic potentials of NiO nanoparticles on NRK-52E kidney epithelial cells. The following assays were used: the nanoparticle characterization by transmission electron microscopy (TEM) and dynamic light scattering (DLS); the determination of cellular uptake and morphologic changes by TEM and inductively coupled plasma-mass spectrometry (ICP-MS); MTT and neutral red uptake (NRU) assays for cytotoxicity; comet assay for genotoxicity; the determination of malondialdehyde (MDA), 8-hydroxydeoxyguanosine (8-OHdG), protein carbonyl (PC) and glutathione (GSH) levels by enzyme-linked immune sorbent assays (ELISA) for the potential of oxidative damage; and Annexin V-FITC apoptosis detection assay with propidium iodide (PI) for apoptosis. The nanoparticles were taken up by the cells and induced dose-dependent DNA damage by comet assay and oxidative damage evidenced by increasing levels of MDA, 8-OHdG, PC and depletion of GSH. At ≥294.0 μg/mL concentration, NiO nanoparticles caused 50% inhibition in cell viability by the cytotoxicity assays. Also, they showed apoptotic/necrotic effects on the cells as well as some morphological changes. We have indicated that their cellular damage effects should raise concern about the safety associated with their applications in consumer products.

  5. Various Forms of Tissue Damage and Danger Signals Following Hematopoietic Stem-Cell Transplantation

    PubMed Central

    Ramadan, Abdulraouf; Paczesny, Sophie

    2014-01-01

    Hematopoietic stem-cell transplantation (HSCT) is the most potent curative therapy for many malignant and non-malignant disorders. Unfortunately, a major complication of HSCT is graft-versus-host disease (GVHD), which is mediated by tissue damage resulting from the conditioning regimens before the transplantation and the alloreaction of dual immune components (activated donor T-cells and recipient’s antigen-presenting cells). This tissue damage leads to the release of alarmins and the triggering of pathogen-recognition receptors that activate the innate immune system and subsequently the adaptive immune system. Alarmins, which are of endogenous origin, together with the exogenous pathogen-associated molecular patterns (PAMPs) elicit similar responses of danger signals and represent the group of damage-associated molecular patterns (DAMPs). Effector cells of innate and adaptive immunity that are activated by PAMPs or alarmins can secrete other alarmins and amplify the immune responses. These complex interactions and loops between alarmins and PAMPs are particularly potent at inducing and then aggravating the GVHD reaction. In this review, we highlight the role of these tissue damaging molecules and their signaling pathways. Interestingly, some DAMPs and PAMPs are organ specific and GVHD-induced and have been shown to be interesting biomarkers. Some of these molecules may represent potential targets for novel therapeutic approaches. PMID:25674088

  6. Chromosomal damage and apoptosis analysis in exfoliated oral epithelial cells from mouthwash and alcohol users

    PubMed Central

    Rocha, Rodrigo dos Santos; Meireles, José Roberto Cardoso; de Moraes Marcílio Cerqueira, Eneida

    2014-01-01

    Chromosomal damage and apoptosis were analyzed in users of mouthwash and/or alcoholic beverages, using the micronucleus test on exfoliated oral mucosa cells. Samples from four groups of 20 individuals each were analyzed: three exposed groups (EG1, EG2 and EG3) and a control group (CG). EG1 comprised mouthwash users; EG2 comprised drinkers, and EG3 users of both mouthwashes and alcoholic beverages. Cell material was collected by gently scraping the insides of the cheeks. Then the cells were fixed in a methanol/acetic acid (3:1) solution and stained and counterstained, respectively, with Schiff reactive and fast green. Endpoints were computed on 2,000 cells in a blind test. Statistical analysis showed that chromosomal damage and apoptosis were significantly higher in individuals of groups EG1 and EG3 than in controls (p < 0.005 and p < 0.001, respectively). No significant difference in chromosomal damage and apoptosis was observed between the exposed groups. In EG2, only the occurrence of apoptosis was significantly higher than in the controls. These results suggest that mouthwashes alone or in association with alcoholic drinks induce genotoxic effects, manifested as chromosomal damage and apoptosis. They also suggest that alcoholic drinks are effective for stimulating the process of apoptosis. However, these data need to be confirmed in larger samples. PMID:25505845

  7. Chronic polycyclic aromatic hydrocarbon exposure causes DNA damage and genomic instability in lung epithelial cells

    PubMed Central

    Bai, Hongzhen; Wu, Min; Zhang, Hongjian; Tang, Guping

    2017-01-01

    Cell exposure to atmospheric polycyclic aromatic hydrocarbons (PAHs) is closely associated with DNA damage and genomic instability. We assessed the mechanisms of chronic and acute PAH exposure-induced genotoxicity in two human lung epithelial cell lines, A549 and NC-H1975. We sampled atmospheric PAHs at the Xixi Campus, Zhejiang University in Hangzhou, China during August (non-haze episode) and November (haze episode) 2015. We identified vehicle emissions as a dominant anthropogenic PAH source in our study. PAHs were extracted according to the United States Environmental Protection Agency Method TO-13A. We found that chronic PAH exposure saturated lung cell xenobiotic metabolic pathways, promoting intercellular reactive oxygen species production and accumulation. Chronic alteration of the cellular redox status resulted in DNA damage and genomic instability. Chronic PAH exposure also perturbed the cellular DNA damage response, inducing S phase arrest and inhibiting apoptosis. Dysregulation of PAH metabolism and the DNA damage response altered cellular homeostasis and increased cell susceptibility to subsequent PAH exposures, thereby enhancing the likelihood of genomic mutation and instability. PMID:29108285

  8. DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A NATURAL BIO-DEFENSE MECHANISM

    EPA Science Inventory

    DNA DAMAGE REPAIR AND CELL CYCLE CONTROL: A natural bio-defense mechanism
    Anuradha Mudipalli.

    Maintenance of genetic information, including the correct sequence of nucleotides in DNA, is essential for replication, gene expression, and protein synthesis. DNA lesions onto...

  9. Comparative DNA damage and transcriptomic effects of engineered nanoparticles in human lung cells in vitro

    EPA Science Inventory

    A series of six titanium dioxide and two cerium oxide engineered nanomaterials were assessed for their ability to induce cytotoxicity, reactive oxygen species (ROS), various types of DNA damage, and transcriptional changes in human respiratory BEAS-2B cells exposed in vitro at se...

  10. Curcumin alters gene expression-associated DNA damage, cell cycle, cell survival and cell migration and invasion in NCI-H460 human lung cancer cells in vitro.

    PubMed

    Chiang, I-Tsang; Wang, Wei-Shu; Liu, Hsin-Chung; Yang, Su-Tso; Tang, Nou-Ying; Chung, Jing-Gung

    2015-10-01

    Lung cancer is the most common cause of cancer mortality and new cases are on the increase worldwide. However, the treatment of lung cancer remains unsatisfactory. Curcumin has been shown to induce cell death in many human cancer cells, including human lung cancer cells. However, the effects of curcumin on genetic mechanisms associated with these actions remain unclear. Curcumin (2 µM) was added to NCI-H460 human lung cancer cells and the cells were incubated for 24 h. Total RNA was extracted from isolated cells for cDNA synthesis, labeling, microarray hybridization and flour‑labeled cDNA hybridized on chip. Localized concentrations of fluorescent molecules were detected and quantified using Expression Console software (Affymetrix) with default RMA parameters. GeneGo software was used for the key genes involved and their possible interaction pathways. The results showed that ~170 genes were significantly upregulated and 577 genes were significantly downregulated in curcumin‑treated cells. Specifically, the up‑ and downregulated genes included CCNE2, associated with DNA damage; ID3, associated with cell survival and 146 genes with a >2- to 3-fold change including the TP53INP1 gene, associated with DNA damage; CDC6, CDCA5, TAKMIP2, CDK14, CDK5, CDCA76, CDC25A, CDC5L and SKP2, associated with cell cycle; the CARD6, ID1 and ID2 genes, associated with cell survival and the BRMS1L, associated with cell migration and invasion. Additionally, 59 downregulated genes exhibited a >4-fold change, including the DDIT3 gene, associated with DNA damage; while 97 genes had a >3- to 4-fold change including the DDIT4 gene, associated with DNA damage; the CCPG1 gene, associated with cell cycle and 321 genes with a >2- to 3-fold including the GADD45A and CGREF1 genes, associated with DNA damage; the CCPG1 gene, associated with cell cycle, the TNFRSF10B, GAS5, TSSC1 and TNFRSF11B gene, associated with cell survival and the ARHAP29 and CADM2 genes, associated with cell migration

  11. LicA induces autophagy through ULK1/Atg13 and ROS pathway in human hepatocellular carcinoma cells.

    PubMed

    Niu, Qiang; Zhao, Wei; Wang, Jin; Li, Chunming; Yan, Tao; Lv, Wei; Wang, Guojing; Duan, Weihong; Zhang, Tao; Wang, Kunnan; Zhou, Dinghua

    2018-05-01

    Chemotherapy is the best choice for the vast majority of hepatocellular carcinoma patients at late stage, but few effective chemotherapy drugs are available in clinic. Licochalcone A (LicA) is a new chemotherapy drug inducing apoptosis as Bcl-2 inhibitor, but few studies report on LicA‑induced autophagy. This study investigated the phenomenon and mechanisms of LicA-induced autophagy looking for a targeted combination drug. Human hepatocellular carcinoma cells (HCCs) were treated with LicA, to detect markers of autophagy and to investigate the mechanisms. In order to investigate the role of reactive oxygen species (ROS) in LicA‑induced autophagy, ROS, glutathione (GSH) and O2- were measured in LicA treated HCCs, and antioxidant N-Acetyl-L-cysteine (NAC) was cotreated with LicA in HCCs, then mechanisms of ROS-induced autophagy was investigated in LicA or LicA combined with NAC treated HCCs. Finally, the LicA-induced apoptosis was detected in LicA combined with NAC treated HCCs. We first report that LicA can induce autophagy through ULK1/Atg13 and ROS pathway in HCCs, suppression of LicA-induced ROS through antioxidant NAC can enhance LicA-induced apoptosis, promoting the function of LicA killing HCCs. LicA can activate the ULK1/Atg13 complex which is upstream of autophagy, additionally, LicA also can promote ROS generation, ROS trigger the expression level of TSC1/2 complex, PRAS40, CTMP, PP2A, PDK1 and Rubicon change, these molecules are upstream of autophagy. In conclusion, LicA can induce autophagy through ULK1/Atg13 and ROS pathway in HCCs, LicA combined with NAC can enhance LicA-induced apoptosis. Our results may provide a novel design for clinical hepatocellular carcinoma therapy trials.

  12. Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells.

    PubMed

    Li, Guang-Yu; Fan, Bin; Ma, Tong-Hui

    2011-01-01

    Visible light has been previously demonstrated to induce retinal ganglion cell (RGC)-5 cell death through the mitochondrial pathway. The present study was designed to determine whether visible light might also directly trigger the death pathway by damaging nuclear DNA. RGC-5 cells were exposed to various intensities and durations of visible light exposure. Cell viability and death were monitored with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide staining. Nuclear DNA damage caused by light was determined with the plasmid assay, genome DNA assay, and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling. The subsequent activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was measured with western blot, and PARP-1's role in the death pathway was assessed by using specific inhibitors. Poly (ADP-ribose) glycohydrolase and apoptosis-inducing factor (AIF) inhibitors were used to show their influence on light-induced cell death. Calcium influx was examined with the fura-2 assay and calcium channel blocker. We found that visible light induced RGC-5 cell death in a time- and intensity-dependent manner. After the light intensity was increased to 2,600 lx, activation of the death pathway in RGC-5 cells was clearly observed by detecting double-strand DNA breaks and nuclear DNA damage in vitro. Nuclear enzyme PARP-1 was promptly activated after exposure to 2,600 lx of light for 2 days, and specific inhibitors of PARP-1 had significant neuroprotective effects. The poly(ADP-ribose) glycohydrolase inhibitor tannic acid and AIF inhibitor N-phenylmaleimide partially protected RGC-5 cells from light injury. A massive calcium influx was detected after 2 days of light exposure, and a calcium channel blocker partially protected cells against light injury. These results suggest that visible light exposure may directly cause nuclear DNA damage, which consequently activates PARP-1. In addition, RGC-5 cells damaged

  13. Visible light may directly induce nuclear DNA damage triggering the death pathway in RGC-5 cells

    PubMed Central

    Fan, Bin; Ma, Tong-Hui

    2011-01-01

    Purpose Visible light has been previously demonstrated to induce retinal ganglion cell (RGC)-5 cell death through the mitochondrial pathway. The present study was designed to determine whether visible light might also directly trigger the death pathway by damaging nuclear DNA. Methods RGC-5 cells were exposed to various intensities and durations of visible light exposure. Cell viability and death were monitored with the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and propidium iodide staining. Nuclear DNA damage caused by light was determined with the plasmid assay, genome DNA assay, and in situ terminal deoxynucleotidyl transferase dUTP nick end labeling. The subsequent activation of nuclear enzyme poly(ADP-ribose) polymerase-1 (PARP-1) was measured with western blot, and PARP-1’s role in the death pathway was assessed by using specific inhibitors. Poly (ADP-ribose) glycohydrolase and apoptosis-inducing factor (AIF) inhibitors were used to show their influence on light-induced cell death. Calcium influx was examined with the fura-2 assay and calcium channel blocker. Results We found that visible light induced RGC-5 cell death in a time- and intensity-dependent manner. After the light intensity was increased to 2,600 lx, activation of the death pathway in RGC-5 cells was clearly observed by detecting double-strand DNA breaks and nuclear DNA damage in vitro. Nuclear enzyme PARP-1 was promptly activated after exposure to 2,600 lx of light for 2 days, and specific inhibitors of PARP-1 had significant neuroprotective effects. The poly(ADP-ribose) glycohydrolase inhibitor tannic acid and AIF inhibitor N-phenylmaleimide partially protected RGC-5 cells from light injury. A massive calcium influx was detected after 2 days of light exposure, and a calcium channel blocker partially protected cells against light injury. Conclusions These results suggest that visible light exposure may directly cause nuclear DNA damage, which consequently activates

  14. Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP)

    PubMed Central

    2014-01-01

    Background Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. Methods Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. Results Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. Conclusions SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples. PMID:24965603

  15. Quantitative measurement of alterations in DNA damage repair (DDR) pathways using single cell network profiling (SCNP).

    PubMed

    Rosen, David B; Leung, Ling Y; Louie, Brent; Cordeiro, James A; Conroy, Andrew; Shapira, Iuliana; Fields, Scott Z; Cesano, Alessandra; Hawtin, Rachael E

    2014-06-25

    Homologous recombination repair (HRR) pathway deficiencies have significant implications for cancer predisposition and treatment strategies. Improved quantitative methods for functionally characterizing these deficiencies are required to accurately identify patients at risk of developing cancer and to identify mechanisms of drug resistance or sensitivity. Flow cytometry-based single cell network profiling (SCNP) was used to measure drug-induced activation of DNA damage response (DDR) proteins in cell lines with defined HRR pathway mutations (including ATM-/-, ATM+/-, BRCA1+/-, BRCA2-/-) and in primary acute myeloid leukemia (AML) samples. Both non-homologous end joining (NHEJ) and HRR pathways were examined by measuring changes in intracellular readouts (including p-H2AX, p-ATM, p-DNA-PKcs, p-53BP1, p-RPA2/32, p-BRCA1, p-p53, and p21) in response to exposure to mechanistically distinct genotoxins. The cell cycle S/G2/M phase CyclinA2 marker was used to normalize for proliferation rates. Etoposide induced proliferation-independent DNA damage and activation of multiple DDR proteins in primary AML cells and ATM +/+but not ATM -/- cell lines. Treatment with the PARPi AZD2281 +/- temozolomide induced DNA damage in CyclinA2+ cells in both primary AML cells and cell lines and distngiushed cell lines deficient (BRCA2-/-) or impaired (BRCA1+/-) in HRR activity from BRCA1+/+ cell lines based on p-H2AX induction. Application of this assay to primary AML samples identified heterogeneous patterns of repair activity including muted or proficient activation of NHEJ and HRR pathways and predominant activation of NHEJ in a subset of samples. SCNP identified functional DDR readouts in both NHEJ and HRR pathways, which can be applied to identify cells with BRCA1+/- haploinsuffiency and characterize differential DDR pathway functionality in primary clinical samples.

  16. Membrane damage effect of continuous wave ultrasound on K562 human leukemia cells.

    PubMed

    Wang, Pan; Li, Yixiang; Wang, Xiaobing; Guo, Ling; Su, Xiaomin; Liu, Quanhong

    2012-12-01

    This study investigated the bioeffects of ultrasound with a frequency of 1.1 MHz on human chronic myelogenous leukemia cell line K562. Membrane potential changes were evaluated by flow cytometry using fluorescent probe bis-(1,3-dibarbituric acid)-trimethine oxanol staining. Other related changes such as potassium ion efflux and intracellular calcium ion overload were also measured. The plasma membrane integrity was monitored by flow cytometry combined with fluorescein diacetate and propidium iodide double fluorescent dye staining. A cell-counting assay and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analysis were used to examine the viability of K562 cells after ultrasound exposure. The acoustic cavitation activity in ultrasound fields was assessed by monitoring hydroxyl radical production. As the ultrasonic intensity increased, the hydroxyl radical produced in the medium increased, and cell membrane damage and cell viability loss were enhanced. The ultrasonic intensity at 0.64 W/cm(2) did not cause substantial cell damage, whereas ultrasound exposure at 1 and 2.1 W/cm(2) could induce serious cell death (14.0% and 40.7%, respectively). Moreover, ultrasound at 0.64 W/cm(2) did not cause substantial membrane potential changes, whereas ultrasound exposure at 1 W/cm(2) could induce depolarization, and fast hyperpolarization occurred when the ultrasonic intensity increased to 2.1 W/cm(2). In addition, compared with control cells, in different ultrasound-treated cells, the potassium ion continuously outflowed with a prolonged incubation time, whereas the intracellular calcium ion oscillations became more apparent. The damaging effects of ultrasound on the cell membrane and cell viability were intensity dependent. The membrane potential changes may be due to acoustic cavitation accompanied by alterations in the balance of ions on opposite sides of the cellular membrane.

  17. Androgen Receptor Accelerates Premature Senescence of Human Dermal Papilla Cells in Association with DNA Damage

    PubMed Central

    Yang, Yi-Chien; Fu, Hung-Chun; Wu, Ching-Yuan; Wei, Kuo-Ting; Huang, Ko-En; Kang, Hong-Yo

    2013-01-01

    The dermal papilla, located in the hair follicle, expresses androgen receptor and plays an important role in hair growth. Androgen/Androgen receptor actions have been implicated in the pathogenesis of androgenetic alopecia, but the exact mechanism is not well known. Recent studies suggest that balding dermal papilla cells exhibit premature senescence, upregulation of p16INK4a, and nuclear expression of DNA damage markers. To investigate whether androgen/AR signaling influences the premature senescence of dermal papilla cells, we first compared frontal scalp dermal papilla cells of androgenetic alopecia patients with matched normal controls and observed that premature senescence is more prominent in the dermal papilla cells of androgenetic alopecia patients. Exposure of androgen induced premature senescence in dermal papilla cells from non-balding frontal and transitional zone of balding scalp follicles but not in beard follicles. Overexpression of the AR promoted androgen-induced premature senescence in association with p16INK4a upregulation, whereas knockdown of the androgen receptor diminished the effects of androgen. An analysis of γ-H2AX expression in response to androgen/androgen receptor signaling suggested that DNA damage contributes to androgen/androgen receptor-accelerated premature senescence. These results define androgen/androgen receptor signaling as an accelerator of premature senescence in dermal papilla cells and suggest that the androgen/androgen receptor-mediated DNA damage-p16INK4a axis is a potential therapeutic target in the treatment of androgenetic alopecia. PMID:24244503

  18. Carboxylated nanodiamonds inhibit γ-irradiation damage of human red blood cells.

    PubMed

    Santacruz-Gomez, K; Silva-Campa, E; Melendrez-Amavizca, R; Teran Arce, F; Mata-Haro, V; Landon, P B; Zhang, C; Pedroza-Montero, M; Lal, R

    2016-04-07

    Nanodiamonds when carboxylated (cNDs) act as reducing agents and hence could limit oxidative damage in biological systems. Gamma (γ)-irradiation of whole blood or its components is required in immunocompetent patients to prevent transfusion-associated graft versus host disease (TA-GVHD). However, γ-irradiation of blood also deoxygenates red blood cells (RBCs) and induces oxidative damage, including abnormalities in cellular membranes and hemolysis. Using atomic force microscopy (AFM) and Raman spectroscopy, we examined the effect of cNDs on γ-irradiation mediated deoxygenation and morphological damage of RBCs. γ-Radiation induced several morphological phenotypes, including stomatocytes, codocytes and echinocytes. While stomatocytes and codocytes are reversibly damaged RBCs, echinocytes are irreversibly damaged. AFM images show significantly fewer echinocytes among cND-treated γ-irradiated RBCs. The Raman spectra of γ-irradiated RBCs had more oxygenated hemoglobin patterns when cND-treated, resembling those of normal, non-irradiated RBCs, compared to the non-cND-treated RBCs. cND inhibited hemoglobin deoxygenation and morphological damage, possibly by neutralizing the free radicals generated during γ-irradiation. Thus cNDs have the therapeutic potential to preserve the quality of stored blood following γ-irradiation.

  19. DNA damage in blood cells exposed to low-level lasers.

    PubMed

    Sergio, Luiz Philippe da Silva; Silva, Ana Paula Almeida da; Amorim, Philipi Freitas; Campos, Vera Maria Araújo; Magalhães, Luis Alexandre Gonçalves; de Paoli, Flavia; de Souza da Fonseca, Adenilson

    2015-04-01

    In regenerative medicine, there are increasing applications of low-level lasers in therapeutic protocols for treatment of diseases in soft and in bone tissues. However, there are doubts about effects on DNA, and an adequate dosimetry could improve the safety of clinical applications of these lasers. This work aimed to evaluate DNA damage in peripheral blood cells of Wistar rats induced by low-level red and infrared lasers at different fluences, powers, and emission modes according to therapeutic protocols. Peripheral blood samples were exposed to lasers and DNA damage was accessed by comet assay. In other experiments, DNA damage was accessed in blood cells by modified comet assay using formamidopyrimidine DNA glycosylase (Fpg) and endonuclease III enzymes. Data show that exposure to low-level red and infrared lasers induce DNA damage depending on fluence, power and emission mode, which are targeted by Fpg and endonuclease III. Oxidative DNA damage should be considered for therapeutic efficacy and patient safety in clinical applications based on low-level red and infrared lasers. © 2015 Wiley Periodicals, Inc.

  20. Identification of Noncanonical Wnt Receptors Required for Wnt-3a-Induced Early Differentiation of Human Neural Stem Cells.

    PubMed

    Bengoa-Vergniory, Nora; Gorroño-Etxebarria, Irantzu; López-Sánchez, Inmaculada; Marra, Michele; Di Chiaro, Pierluigi; Kypta, Robert

    2017-10-01

    Wnt proteins preferentially activate either β-catenin-dependent or β-catenin-independent signals, but the activity of a particular Wnt also depends on cellular context and receptor availability. We previously reported that Wnt-3a induces neural differentiation of human embryonic stem cell-derived neural stem cells (NSCs) in a β-catenin-independent manner by activating a signal involving JNK and the AP-1 family member ATF-2. Here, we report the results of a gene silencing approach to identify the Wnt receptors that mediate this response to Wnt-3a. Silencing of ROR2 increased neuronal differentiation, as measured by expression of the genes DCX, NEUROD1, and NGN1, suggesting ROR2 signals normally prevent differentiation. Silencing of the other Wnt receptors singly did not affect Wnt-3a-induced neuronal differentiation. However, pairwise silencing of ROR1 and FZD4 or FZD5 and of LRP6 and FZD4 or FZD5 inhibited neuronal differentiation, as detected by reductions in the expression of neuronal genes and immunocytochemical detection of DCX, NEUROD1 and DCX. Ectopic expression of these receptors in HEK 293 cells increased ATF2-dependent transcription. In addition, ROR1 coimmunoprecipitated with FZD4 and LRP6 in transfected HEK 293 cells and colocalized with FZD4 and with LRP6 at the cell surface of transfected L cells. Wnt-3a did not appear to affect these interactions but did alter the interactions between LRP6 and FZD4/5. Together, these observations highlight roles for ROR1, LRP6, FZD4, and FZD5 in neural stem cell differentiation and provide support for a model in which dynamic interactions among these receptors mediate Wnt-3a activation of ATF2 signaling.

  1. Inflammation-Induced Cell Proliferation Potentiates DNA Damage-Induced Mutations In Vivo

    PubMed Central

    Kiraly, Orsolya; Gong, Guanyu; Olipitz, Werner; Muthupalani, Sureshkumar; Engelward, Bevin P.

    2015-01-01

    Mutations are a critical driver of cancer initiation. While extensive studies have focused on exposure-induced mutations, few studies have explored the importance of tissue physiology as a modulator of mutation susceptibility in vivo. Of particular interest is inflammation, a known cancer risk factor relevant to chronic inflammatory diseases and pathogen-induced inflammation. Here, we used the fluorescent yellow direct repeat (FYDR) mice that harbor a reporter to detect misalignments during homologous recombination (HR), an important class of mutations. FYDR mice were exposed to cerulein, a potent inducer of pancreatic inflammation. We show that inflammation induces DSBs (γH2AX foci) and that several days later there is an increase in cell proliferation. While isolated bouts of inflammation did not induce HR, overlap between inflammation-induced DNA damage and inflammation-induced cell proliferation induced HR significantly. To study exogenously-induced DNA damage, animals were exposed to methylnitrosourea, a model alkylating agent that creates DNA lesions relevant to both environmental exposures and cancer chemotherapy. We found that exposure to alkylation damage induces HR, and importantly, that inflammation-induced cell proliferation and alkylation induce HR in a synergistic fashion. Taken together, these results show that, during an acute bout of inflammation, there is a kinetic barrier separating DNA damage from cell proliferation that protects against mutations, and that inflammation-induced cell proliferation greatly potentiates exposure-induced mutations. These studies demonstrate a fundamental mechanism by which inflammation can act synergistically with DNA damage to induce mutations that drive cancer and cancer recurrence. PMID:25647331

  2. Induction of ROS Overload by Alantolactone Prompts Oxidative DNA Damage and Apoptosis in Colorectal Cancer Cells.

    PubMed

    Ding, Yushuang; Wang, Hongge; Niu, Jiajing; Luo, Manyu; Gou, Yangmei; Miao, Lining; Zou, Zhihua; Cheng, Ying

    2016-04-14

    Cancer cells typically display higher than normal levels of reactive oxygen species (ROS), which may promote cancer development and progression but may also render the cancer cells more vulnerable to further ROS insult. Indeed, many of the current anticancer therapeutics kill cancer cells via induction of oxidative stress, though they target both cancer and normal cells. Recently, alantolactone (ATL), a natural sesquiterpene lactone, has been shown to induce apoptosis by increasing ROS levels specifically in cancer cells; however, the molecular mechanisms linking ROS overproduction to apoptosis remain unclear. Here we show that the ATL-induced ROS overload in human SW480 and SW1116 colorectal cancer cells was followed by a prominent accumulation of cellular oxidized guanine (8-oxoG) and immediate increase in the number of DNA strand breaks, indicating that increased ROS resulted in extensive oxidative DNA damage. Consequently, the G₁/S-CDK suppresser CDKN1B (p21) and pro-apoptotic proteins Bax and activated caspase-3 were upregulated, while anti-apoptotic Bcl-2 was downregulated, which were followed by cell cycle arrest at G₁ and marked apoptosis in ATL-treated cancer but not non-cancer cells. These results suggest that the ATL-induced ROS overload triggers cell death through induction of massive oxidative DNA damage and subsequent activation of the intrinsic apoptosis pathway.

  3. Flavivirus NS4A-induced Autophagy Protects Cells against Death and Enhances Virus Replication*

    PubMed Central

    McLean, Jeffrey E.; Wudzinska, Aleksandra; Datan, Emmanuel; Quaglino, Daniela; Zakeri, Zahra

    2011-01-01

    Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication. PMID:21511946

  4. Flavivirus NS4A-induced autophagy protects cells against death and enhances virus replication.

    PubMed

    McLean, Jeffrey E; Wudzinska, Aleksandra; Datan, Emmanuel; Quaglino, Daniela; Zakeri, Zahra

    2011-06-24

    Flaviviruses include the most prevalent and medically challenging viruses. Persistent infection with flaviviruses of epithelial cells and hepatocytes that do not undergo cell death is common. Here, we report that, in epithelial cells, up-regulation of autophagy following flavivirus infection markedly enhances virus replication and that one flavivirus gene, NS4A, uniquely determines the up-regulation of autophagy. Dengue-2 and Modoc (a murine flavivirus) kill primary murine macrophages but protect epithelial cells and fibroblasts against death provoked by several insults. The flavivirus-induced protection derives from the up-regulation of autophagy, as up-regulation of autophagy by starvation or inactivation of mammalian target of rapamycin also protects the cells against insult, whereas inhibition of autophagy via inactivation of PI3K nullifies the protection conferred by flavivirus. Inhibition of autophagy also limits replication of both Dengue-2 and Modoc virus in epithelial cells. Expression of flavivirus NS4A is sufficient to induce PI3K-dependent autophagy and to protect cells against death; expression of other viral genes, including NS2A and NS4B, fails to protect cells against several stressors. Flavivirus NS4A protein induces autophagy in epithelial cells and thus protects them from death during infection. As autophagy is vital to flavivirus replication in these cells, NS4A is therefore also identified as a critical determinant of flavivirus replication.

  5. Wnt5a attenuates Wnt3a-induced alkaline phosphatase expression in dental follicle cells

    SciTech Connect

    Sakisaka, Yukihiko; Tsuchiya, Masahiro; Tohoku Fukushi University, Sendai 989-3201

    2015-08-01

    Wnt signaling regulates multiple cellular events such as cell proliferation, differentiation, and apoptosis through β-catenin-dependent canonical and β-catenin-independent noncanonical pathways. Canonical Wnt/β-catenin signaling can promote the differentiation of dental follicle cells, putative progenitor cells for cementoblasts, osteoblasts, and periodontal ligament cells, toward a cementoblast/osteoblast phenotype during root formation, but little is known about the biological significance of noncanonical Wnt signaling in this process. We identified the expression of Wnt5a, a representative noncanonical Wnt ligand, in tooth root lining cells (i.e. precementoblasts/cementoblasts) and dental follicle cells during mouse tooth root development, as assessed by immunohistochemistry. Silencing expression of the Wnt5a genemore » in a dental follicle cell line resulted in enhancement of the Wnt3a (a representative canonical Wnt ligand)-mediated increase in alkaline phosphatase (ALP) expression. Conversely, treatment with recombinant Wnt5a inhibited the increase in ALP expression, suggesting that Wnt5a signaling functions as a negative regulator of canonical Wnt-mediated ALP expression of dental follicle cells. Wnt5a did not affect the nuclear translocation of β-catenin as well as β-catenin-mediated transcriptional activation of T-cell factor (Tcf) triggered by Wnt3a, suggesting that Wnt5a inhibits the downstream part of the β-catenin-Tcf pathway. These findings suggest the existence of a feedback mechanism between canonical and noncanonical Wnt signaling during the differentiation of dental follicle cells. - Highlights: • Dental follicle cells express Wnt5a during tooth root development. • Silencing of Wnt5a enhances Wnt3a-mediated ALP expression of dental follicle cells. • Conversely, treatment with rWnt5a inhibited the increase in ALP expression. • Wnt5a functions as a negative regulator of Wnt3a-mediated ALP expression.« less

  6. A thermochemical model of radiation damage and annealing applied to GaAs solar cells

    NASA Technical Reports Server (NTRS)

    Conway, E. J.; Walker, G. H.; Heinbockel, J. H.

    1981-01-01

    Calculations of the equilibrium conditions for continuous radiation damage and thermal annealing are reported. The calculations are based on a thermochemical model developed to analyze the incorporation of point imperfections in GaAs, and modified by introducing the radiation to produce native lattice defects rather than high-temperature and arsenic atmospheric pressure. The concentration of a set of defects, including vacancies, divacancies, and impurity vacancy complexes, are calculated as a function of temperature. Minority carrier lifetimes, short circuit current, and efficiency are deduced for a range of equilibrium temperatures. The results indicate that GaAs solar cells could have a mission life which is not greatly limited by radiation damage.

  7. Immune response involved in liver damage and the activation of hepatic progenitor cells during liver tumorigenesis.

    PubMed

    Hou, Xiao-Juan; Ye, Fei; Li, Xiao-Yong; Liu, Wen-Ting; Jing, Ying-Ying; Han, Zhi-Peng; Wei, Li-Xin

    2018-04-01

    Hepatocellular carcinoma (HCC) is a typical inflammation-related cancer. Hepatitis B virus (HBV) and hepatitis C virus (HCV) infection are well-known leading causes of HCC. However, the mechanism of the induction of HCC by these virus is still being debated. This review will focus on the current knowledge of the pathogenesis of HBV- and HCV-induced inflammation and the role of such immune activation in the tumorigenesis of HCC. It is well established that the recruitment of certain number and type of immune cells to liver is essential for the resolution of HBV and HCV infection and the prevention of subsequent chronic persistent infection. However, in case that the immune response do not completely clear virus, persistent chronic infection occurs, and the perpetual immune response may contribute to chronic damages of the liver. Such chronic inflammatory damages further harm hepatocytes, but not hepatic progenitor cells (HPCs). Thus, following chronic damages, HPCs are activated and their dysregulated proliferation ensures survival in the hostile environment, contributing to the tumorigenesis of HCC. Furthermore, accumulating evidence also provides a strong link between HPCs and human hepatocellular carcinoma. Collectively, these findings support a notion that immune response is involved in liver damage during hepatitis virus infection, and the activation and dysregulated differentiation of hepatic progenitor cells promote the tumorigenesis of human hepatocellular carcinoma. Copyright © 2017. Published by Elsevier Inc.

  8. Damaging effects of water-borne cadmium chloride on DNA of lung cells of immature mice.

    PubMed

    Yang, X F; Ge, Y M; Zhang, H T; Ning, H M; Jiang, J Q; Qi, Y H; Wang, Z L

    2012-12-17

    We investigated the effects of cadmium on lung cell DNA in immature mice. The mice were randomly divided into four groups: control group, low-dose group (1/100 LD(50)), middle-dose group (1/50 LD(50)), and high-dose group (1/25 LD(50)); they were supplied with cadmium chloride or control water for 40 days. Lung cells collected from sacrificed mice were used to evaluate the extent of DNA damage by comet assay. The ratio of tailing cells, DNA tail length, DNA comet length, DNA tail moment, DNA olive tail moment, and percentage of DNA in the comet tail were measured. The rate of tailing lung cells exposed to cadmium increased significantly; the low-concentration group had significantly (P < 0.05) higher rates, and the middle- and high-concentration groups had higher (P < 0.01) rates compared to the control. DNA tail length, DNA comet length, DNA tail moment, and DNA olive tail moment all increased with the increase in cadmium doses, but compared with those of the control group, no significant differences in low-dose group were found (P > 0.05), and the differences in middle- and high-dose groups were all highly significant (P < 0.01). The degree of DNA damage also increased with the increase of the cadmium concentrations. We conclude that cadmium significantly increases DNA damage in lung cells of immature mice in a dose-dependent manner.

  9. Splicing function of mitotic regulators links R-loop–mediated DNA damage to tumor cell killing

    PubMed Central

    Wan, Yihan; Zheng, Xiaobin; Chen, Haiyang; Guo, Yuxuan; Jiang, Hao; He, Xiaonan

    2015-01-01

    Although studies suggest that perturbing mitotic progression leads to DNA damage and p53 activation, which in turn lead to either cell apoptosis or senescence, it remains unclear how mitotic defects trigger p53 activation. We show that BuGZ and Bub3, which are two mitotic regulators localized in the interphase nucleus, interact with the splicing machinery and are required for pre-mRNA splicing. Similar to inhibition of RNA splicing by pladienolide B, depletion of either BuGZ or Bub3 led to increased formation of RNA–DNA hybrids (R-loops), which led to DNA damage and p53 activation in both human tumor cells and primary cells. Thus, R-loop–mediated DNA damage and p53 activation offer a mechanistic explanation for apoptosis of cancer cells and senescence of primary cells upon disruption of the dual-function mitotic regulators. This demonstrates the importance of understanding the full range of functions of mitotic regulators to develop antitumor drugs. PMID:25918225

  10. Edaravone ameliorates compression-induced damage in rat nucleus pulposus cells.

    PubMed

    Lin, Hui; Ma, Xuan; Wang, Bai-Chuan; Zhao, Lei; Liu, Jian-Xiang; Pu, Fei-Fei; Hu, Yi-Qiang; Hu, Hong-Zhi; Shao, Zeng-Wu

    2017-11-15

    Edaravone is a strong free radical scavenger most used for treating acute ischemic stroke. In this study we investigated the protective effects and underlying mechanisms of edaravone on compression-induced damage in rat nucleus pulposus (NP) cells. Cell viability was determined using MTT assay methods. NP cell apoptosis was measured by Hoechst 33,258 staining and Annexin V/PI double staining. Intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and intracellular calcium ([Ca 2+ ] i ) were determined by fluorescent probes DCFH-DA, JC-1 and Fluo-3/AM, respectively. Apoptosis-related proteins (cleaved caspase-3, cytosolic cytochrome c, Bax and Bcl-2) and extracellular matrix proteins (aggrecan and collagen II) were analyzed by western blot. Edaravone attenuated the compression-induced decrease in viability of NP cells in a dose-dependent manner. 33,258 and Annexin V/PI double staining showed that edaravone protected NP cells from compression-induced apoptosis. Further studies confirmed that edaravone protected NP cells against compression-induced mitochondrial pathway of apoptosis by inhibiting overproduction of ROS, collapse of MMP and overload of [Ca 2+ ] i . In addition, edaravone promoted the expression of aggrecan and collagen II in compression-treated NP cells. These results strongly indicate that edaravone ameliorates compression-induced damage in rat nucleus pulposus cells. Edaravone could be a potential new drug for treatment of IDD. Copyright © 2017 Elsevier Inc. All rights reserved.

  11. Deficiency in DNA damage response of enterocytes accelerates intestinal stem cell aging inDrosophila.

    PubMed

    Park, Joung-Sun; Jeon, Ho-Jun; Pyo, Jung-Hoon; Kim, Young-Shin; Yoo, Mi-Ae

    2018-03-07

    Stem cell dysfunction is closely linked to tissue and organismal aging and age-related diseases, and heavily influenced by the niche cells' environment. The DNA damage response (DDR) is a key pathway for tissue degeneration and organismal aging; however, the precise protective role of DDR in stem cell/niche aging is unclear. The Drosophila midgut is an excellent model to study the biology of stem cell/niche aging because of its easy genetic manipulation and its short lifespan. Here, we showed that deficiency of DDR in Drosophila enterocytes (ECs) accelerates intestinal stem cell (ISC) aging. We generated flies with knockdown of Mre11 , Rad50 , Nbs1 , ATM , ATR , Chk1 , and Chk2 , which decrease the DDR system in ECs. EC-specific DDR depletion induced EC death, accelerated the aging of ISCs, as evidenced by ISC hyperproliferation, DNA damage accumulation, and increased centrosome amplification, and affected the adult fly's survival. Our data indicated a distinct effect of DDR depletion in stem or niche cells on tissue-resident stem cell proliferation. Our findings provide evidence of the essential role of DDR in protecting EC against ISC aging, thus providing a better understanding of the molecular mechanisms of stem cell/niche aging.

  12. Casuarinin protects cultured MDCK cells from hydrogen peroxide-induced oxidative stress and DNA oxidative damage.

    PubMed

    Chen, Ching-Hsein; Liu, Tsan-Zon; Kuo, Tsun-Cheng; Lu, Fung-Jou; Chen, Yu-Chin; Chang-Chien, Yi-Wen; Lin, Chun-Ching

    2004-11-01

    Casuarinin has been shown to be an antioxidant in acellular experiments. This study was designed to assess the ability of casuarinin, extracted from Terminalia arjuna, to protect cultured Madin-Darby canine kidney (MDCK) cells against H2O2-mediated oxidative stress. A comparison with trolox, a hydrosoluble vitamin E analogue was performed. MDCK cells were pretreated with casuarinin or trolox for 1 h, then exposed to H2O2. After incubation with 0.8 mM H2O2 for 1 h, casuarinin caused a decrease in intracellular peroxide production as shown by dichlorofluorescein (DCF) fluorescence in a concentration-dependent manner. After 3 h exposure to 8 mM H2O2, the percentage of intracellular glutathione (GSH)-negative cells was reduced in the casuarinin-treated group. Addition of 32mM H2O2 to MDCK cells for 3 h induced an increase in the percentage of cells containing 8-oxoguanine but the level of such cells declined in casuarinin-treated cells. These results show that casuarinin is more effective against H2O2-induced oxidative damage than trolox. The data suggest that casuarinin attenuates H2O2-induced oxidative stress, decreases DNA oxidative damage and prevents the depletion of intracellular GSH in MDCK cells.

  13. Differential Response of Mouse Thymic Epithelial Cell Types to Ionizing Radiation-Induced DNA Damage

    PubMed Central

    Calvo-Asensio, Irene; Barthlott, Thomas; von Muenchow, Lilly; Lowndes, Noel F.; Ceredig, Rhodri

    2017-01-01

    Thymic epithelial cells (TECs) are the main components of the thymic stroma that support and control T-cell development. Preparative regimens using DNA-damaging agents, such as total body irradiation and/or chemotherapeutic drugs, that are necessary prior to bone marrow transplantation (BMT) have profound deleterious effects on the hematopoietic system, including the thymic stroma, which may be one of the main causes for the prolonged periods of T-cell deficiency and the inefficient T cell reconstitution that are common following BMT. The DNA damage response (DDR) is a complex signaling network that allows cells to respond to all sorts of genotoxic insults. Hypoxia is known to modulate the DDR and play a role affecting the survival capacity of different cell types. In this study, we have characterized in detail the DDR of cortical and medullary TEC lines and their response to ionizing radiation, as well as the effects of hypoxia on their DDR. Although both mTECs and cTECs display relatively high radio-resistance, mTEC cells have an increased survival capacity to ionizing radiation (IR)-induced DNA damage, and hypoxia specifically decreases the radio-resistance of mTECs by upregulating the expression of the pro-apoptotic factor Bim. Analysis of the expression of TEC functional factors by primary mouse TECs showed a marked decrease of highly important genes for TEC function and confirmed cTECs as the most affected cell type by IR. These findings have important implications for improving the outcomes of BMT and promoting successful T cell reconstitution. PMID:28450862

  14. [Nickel exposure to A549 cell damage and L-ascorbic acid interference effect].

    PubMed

    Fu, Yao; Wang, Yue; Dan, Han; Zhang, Lin; Ma, Wenhan; Pan, Yulin; Wu, Yonghui

    2015-05-01

    Studying different concentrations of nickel smelting smoke subjects of human lung adenocarcinoma cells (A549) carcinogenic effects, discusses the influence of L-ascorbic acid protection. The A549 cells were divided into experimental and L-ascorbic acid in the intervention group. Plus exposure group concentration of nickel refining dusts were formulated 0.00, 6.25, 12.50, 25.00, 50.00, 100.00 µg/ml suspension, the intervention group on the basis of the added exposure group containing L-ascorbic acid (100 mmol/L), contact 24 h. Detection of cell viability by MTT assay. When the test substance concentration select 0.00, 25.00, 50.00, 100.00 µg/ml experiment for internal Flou-3 fluorescent probe to detect cell Ca²⁺ concentration, within DCFH-DA detect intracellular reactive oxygen (ROS) content, real-time quantitative PCR (real time, in the RT-PCR) was used to detect cell HIF-1α gene expression. With the increase of concentration, subjects increased cell growth inhibition rate, intracellular Ca²⁺ concentration increases, ROS content increased, HIF-1α gene expression increased, differences were statistically significant (P < 0.05). After L-ascorbic acid intervention treatment, the results of the intervention group were lower than that of the experimental group, and the difference was statistically significant (P < 0.05), so L-ascorbic acid can effectively protect the nickel exposure damage to cells. With subjects following exposure to nickel concentration increased, its effect on A549 cell damage increases, L-ascorbic acid cell damage caused by nickel has certain protective effect.

  15. Upregulation of GADD45α in light-damaged retinal pigment epithelial cells

    PubMed Central

    Gao, M-L; Deng, W-L; Huang, N; Wang, Y-Y; Lei, X-L; Xu, Z-Q; Hu, D-N; Cai, J-Q; Lu, F; Jin, Z-B

    2016-01-01

    To better understand the molecular mechanisms responsible for light-induced damage in retinal pigmented epithelial (RPE) cells, we developed an automated device to recapitulate intense light exposure. When compared with human fibroblasts, ARPE-19 cells that had been exposed to blue-rich light-emitting diode-light of 10 000 Lux at 37 °C for 9 h displayed dramatic cellular apoptosis. Collectively, gene expression profiling and qPCR demonstrated that growth arrest and DNA damage-45α (GADD45α) expression was markedly upregulated. Transient knockdown of GADD45α partially attenuated light-damage-induced apoptosis in ARPE-19 cells, whereas GADD45α overexpression dramatically increased it. These results demonstrate the critical function of GADD45α in light-induced RPE cellular apoptosis. Quantitative reverse transcription-PCR and western blotting revealed that the upregulation of GADD45α was under direct control of p53. Moreover, treatment with Ly294002, an inhibitor of AKT phosphorylation, further promoted GADD45α gene transcription in both non-light and light-damaged ARPE-19 cells. Treatment also exacerbated RPE cellular apoptosis after light exposure, confirming that inhibition of Akt phosphorylation increases GADD45α expression. Collectively, our findings reveal that light irrigation induces human RPE cellular apoptosis through upregulation of GADD45α expression mediated through both the p53 and phosphatidylinositol 3-kinase-AKT signaling pathways. These results provide new insights into human retinal diseases elicited by light damage and open a new avenue for disease prevention and treatment. PMID:27551507

  16. DNA damage mediated s and g(2) checkpoints in human embryonal carcinoma cells.

    PubMed

    Wang, XiaoQi; Lui, Vincent C H; Poon, Ronnie T P; Lu, Ping; Poon, Randy Y C

    2009-03-01

    For mouse embryonic stem (ES) cells, the importance of the S and G(2) cell cycle checkpoints for genomic integrity is increased by the absence of the G(1) checkpoint. We have investigated ionizing radiation (IR)-mediated cell cycle checkpoints in undifferentiated and retinoic acid-differentiated human embryonal carcinoma (EC) cells. Like mouse ES cells, human EC cells did not undergo G(1) arrest after IR but displayed a prominent S-phase delay followed by a G(2)-phase delay. In contrast, although differentiated EC cells also failed to arrest at G(1)-phase after IR, they quickly exited S-phase and arrested in G(2)-phase. In differentiated EC cells, the G(2)-M-phase cyclin B1/CDC2 complex was upregulated after IR, but the G(1)-S-phase cyclin E and the cyclin E/CDK2 complex were expressed at constitutively low levels, which could be an important factor distinguishing DNA damage responses between undifferentiated and differentiated EC cells. S-phase arrest and expression of p21 could be inhibited by 7-hydroxystaurosporine, suggesting that the ataxia-telangiectasia and Rad-3-related-checkpoint kinase 1 (ATR-CHK1), and p21 pathways might play a role in the IR-mediated S-phase checkpoint in EC cells. IR-mediated phosphorylation of ataxia-telangiectasia mutated, (CHK1), and checkpoint kinase 2 were distinctly higher in undifferentiated EC cells compared with differentiated EC cells. Combined with the prominent S and G(2) checkpoints and a more efficient DNA damage repair system, these mechanisms operate together in the maintenance of genome stability for EC cells.

  17. EdU induces DNA damage response and cell death in mESC in culture.

    PubMed

    Kohlmeier, Fanni; Maya-Mendoza, Apolinar; Jackson, Dean A

    2013-03-01

    Recently, a novel DNA replication precursor analogue called 5-ethynyl-2'-deoxyuridine (EdU) has been widely used to monitor DNA synthesis as an alternative to bromodeoxyuridine. Use of EdU benefits from simplicity and reproducibility and the simple chemical detection systems allows excellent preservation of nuclear structure. However, the alkyne moiety is highly reactive, raising the possibility that incorporation might compromise genome stability. To assess the extent of possible DNA damage, we have analysed the effect of EdU incorporation into DNA during short- and long-term cell culture using a variety of cell lines. We show that EdU incorporation has no measurable impact on the rate of elongation of replication forks during synthesis. However, using different cell lines we find that during long-term cell culture variable responses to EdU incorporation are seen, which range from delayed cell cycle progression to complete cell cycle arrest. The most profound phenotypes were seen in mouse embryonic stem cells, which following incorporation of EdU accumulated in the G2/M-phase of the cell cycle before undergoing apoptosis. In long-term cell culture, EdU incorporation also triggered a DNA damage response in all cell types analysed. Our study shows that while EdU is extremely useful to tag sites of on-going replication, for long-term studies (i.e. beyond the cell cycle in which labelling is performed), a careful analysis of cell cycle perturbations must be performed in order to ensure that any conclusions made after EdU treatment are not a direct consequence of EdU-dependent activation of cell stress responses.

  18. The use of microbeams to investigate radiation damage in living cells

    PubMed Central

    Folkard, Melvyn; Prise, Kevin M.; Grime, Geoff; Kirkby, Karen; Vojnovic, Borivoj

    2010-01-01

    The micro-irradiation technique continues to be highly relevant to a number of radiobiological studies in vitro. In particular, studies of the bystander effect show that direct damage to cells is not the only trigger for radiation-induced effects, but that unirradiated cells can also respond to signals from irradiated neighbours. Furthermore, the bystander response can be initiated even when no energy is deposited in the genomic DNA of the irradiated cell (i.e. by targeting just the cytoplasm). PMID:18710814

  19. Cell membrane damage is involved in the impaired survival of bone marrow stem cells by oxidized low-density lipoprotein

    PubMed Central

    Li, Xin; Xiao, Yuan; Cui, Yuqi; Tan, Tao; Narasimhulu, Chandrakala A; Hao, Hong; Liu, Lingjuan; Zhang, Jia; He, Guanglong; Verfaillie, Catherine M; Lei, Minxiang; Parthasarathy, Sampath; Ma, Jianjie; Zhu, Hua; Liu, Zhenguo

    2014-01-01

    Cell therapy with bone marrow stem cells (BMSCs) remains a viable option for tissue repair and regeneration. A major challenge for cell therapy is the limited cell survival after implantation. This study was to investigate the effect of oxidized low-density lipoprotein (ox-LDL, naturally present in human blood) on BMSC injury and the effect of MG53, a tissue repair protein, for the improvement of stem cell survival. Rat bone marrow multipotent adult progenitor cells (MAPCs) were treated with ox-LDL, which caused significant cell death as reflected by the increased LDH release to the media. Exposure of MAPCs to ox-LDL led to entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated reactive oxygen species (ROS) as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death. When MAPCs were treated with the recombinant human MG53 protein (rhMG53) ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. In the presence of rhMG53, the MAPCs showed enhanced cell survival and proliferation. Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs. rhMG53 treatment protected MAPCs against membrane damage and enhanced their survival which might represent a novel means for improving efficacy for stem cell-based therapy for treatment of diseases, especially in setting of hyperlipidemia. PMID:25256620

  20. Improvement and extension of data from ATS-6 Solar Cell Radiation Damage Experiment (SCRDE)

    NASA Technical Reports Server (NTRS)

    Goldhammer, L. J.

    1979-01-01

    The ATS 6 solar cell radiation damage experiment data through 2 1/3 years of synchronous orbit operation are presented. Comparisons are made of the performances of the 13 different types of solar cell/cover configurations, including solar cell and cover thickness variations, base resistivity variation, new cover processes and materials, and the COMSAT violet cell. These performances are also compared to the performance of the LES 6 solar cell experiment, the ATS 6 main solar arrays, and laboratory spectrum electron irradiations. It is found that the cells of the ATS 6 experiment generally performed as expected through 6 to 9 months in orbit, but that at 2 1/3 years they were severely degraded in current. The short circuit current degradation after 2 1/3 years in orbit appears to exhibit an anomalous additional degradation of 5 to 9 percent over what was experienced in synchronous orbit operation.

  1. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival

    PubMed Central

    Tinkum, Kelsey L.; Stemler, Kristina M.; White, Lynn S.; Loza, Andrew J.; Jeter-Jones, Sabrina; Michalski, Basia M.; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S.; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-01-01

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy. PMID:26644583

  2. Fasting protects mice from lethal DNA damage by promoting small intestinal epithelial stem cell survival.

    PubMed

    Tinkum, Kelsey L; Stemler, Kristina M; White, Lynn S; Loza, Andrew J; Jeter-Jones, Sabrina; Michalski, Basia M; Kuzmicki, Catherine; Pless, Robert; Stappenbeck, Thaddeus S; Piwnica-Worms, David; Piwnica-Worms, Helen

    2015-12-22

    Short-term fasting protects mice from lethal doses of chemotherapy through undetermined mechanisms. Herein, we demonstrate that fasting preserves small intestinal (SI) architecture by maintaining SI stem cell viability and SI barrier function following exposure to high-dose etoposide. Nearly all SI stem cells were lost in fed mice, whereas fasting promoted sufficient SI stem cell survival to preserve SI integrity after etoposide treatment. Lineage tracing demonstrated that multiple SI stem cell populations, marked by Lgr5, Bmi1, or HopX expression, contributed to fasting-induced survival. DNA repair and DNA damage response genes were elevated in SI stem/progenitor cells of fasted etoposide-treated mice, which importantly correlated with faster resolution of DNA double-strand breaks and less apoptosis. Thus, fasting preserved SI stem cell viability as well as SI architecture and barrier function suggesting that fasting may reduce host toxicity in patients undergoing dose intensive chemotherapy.

  3. Synaptically silent sensory hair cells in zebrafish are recruited after damage.

    PubMed

    Zhang, Qiuxiang; Li, Suna; Wong, Hiu-Tung C; He, Xinyi J; Beirl, Alisha; Petralia, Ronald S; Wang, Ya-Xian; Kindt, Katie S

    2018-04-11

    Analysis of mechanotransduction among ensembles of sensory hair cells in vivo is challenging in many species. To overcome this challenge, we used optical indicators to investigate mechanotransduction among collections of hair cells in intact zebrafish. Our imaging reveals a previously undiscovered disconnect between hair-cell mechanosensation and synaptic transmission. We show that saturating mechanical stimuli able to open mechanically gated channels are unexpectedly insufficient to evoke vesicle fusion in the majority of hair cells. Although synaptically silent, latent hair cells can be rapidly recruited after damage, demonstrating that they are synaptically competent. Therefore synaptically silent hair cells may be an important reserve that acts to maintain sensory function. Our results demonstrate a previously unidentified level of complexity in sculpting sensory transmission from the periphery.

  4. Modeling femtosecond pulse laser damage on conductors using Particle-In-Cell simulations

    NASA Astrophysics Data System (ADS)

    Mitchell, Robert A.; Schumacher, Douglass; Chowdhury, Enam

    2013-11-01

    We present, to our knowledge, the first adaptation of the Particle-In-Cell (PIC) simulation method for use in the study of femtosecond pulse laser damage, including the first implementation of the Morse potential for PIC codes. We compare the PIC method to a wide variety of currently used modeling schemes, ranging from purely ab-initio molecular dynamics simulations to semi-empirical models with many fitting parameters, and show how PIC simulations can provide a complementary approach by filling the gap in theoretical methodology between the two cases. We detail the necessity and implementation of an inter-atomic pair-potential in PIC studies of laser damage. Lastly, we use our model to treat the full laser damage process of a copper target, and show that our results compare well to simple scaling laws for crater size.

  5. Modeling femtosecond pulse laser damage using particle-in-cell simulations

    NASA Astrophysics Data System (ADS)

    Mitchell, Robert A.; Schumacher, Douglass; Chowdhury, Enam

    2014-12-01

    We present, to our knowledge, the first adaptation of the particle-in-cell (PIC) simulation method for use in the study of femtosecond pulse laser damage, including the first implementation of the Morse pair-potential for PIC codes. We compare the PIC method to a wide variety of currently used modeling schemes, ranging from purely ab initio molecular dynamics simulations to semi-empirical models with many fitting parameters and show how PIC simulations can provide a complementary approach by filling the gap in theoretical methodology between the two cases. We detail the necessity and implementation of an interatomic pair-potential in PIC studies of laser damage. Finally, we use our model to treat the full laser damage process of a copper target and show that our results compare well to simple scaling laws for crater size.

  6. Signalling of DNA damage and cytokines across cell barriers exposed to nanoparticles depends on barrier thickness

    NASA Astrophysics Data System (ADS)

    Sood, A.; Salih, S.; Roh, D.; Lacharme-Lora, L.; Parry, M.; Hardiman, B.; Keehan, R.; Grummer, R.; Winterhager, E.; Gokhale, P. J.; Andrews, P. W.; Abbott, C.; Forbes, K.; Westwood, M.; Aplin, J. D.; Ingham, E.; Papageorgiou, I.; Berry, M.; Liu, J.; Dick, A. D.; Garland, R. J.; Williams, N.; Singh, R.; Simon, A. K.; Lewis, M.; Ham, J.; Roger, L.; Baird, D. M.; Crompton, L. A.; Caldwell, M. A.; Swalwell, H.; Birch-Machin, M.; Lopez-Castejon, G.; Randall, A.; Lin, H.; Suleiman, M.-S.; Evans, W. H.; Newson, R.; Case, C. P.

    2011-12-01

    The use of nanoparticles in medicine is ever increasing, and it is important to understand their targeted and non-targeted effects. We have previously shown that nanoparticles can cause DNA damage to cells cultured below a cellular barrier without crossing this barrier. Here, we show that this indirect DNA damage depends on the thickness of the cellular barrier, and it is mediated by signalling through gap junction proteins following the generation of mitochondrial free radicals. Indirect damage was seen across both trophoblast and corneal barriers. Signalling, including cytokine release, occurred only across bilayer and multilayer barriers, but not across monolayer barriers. Indirect toxicity was also observed in mice and using ex vivo explants of the human placenta. If the importance of barrier thickness in signalling is a general feature for all types of barriers, our results may offer a principle with which to limit the adverse effects of nanoparticle exposure and offer new therapeutic approaches.

  7. Low doses of ionizing radiation to mammalian cells may rather control than cause DNA damage

    SciTech Connect

    Feinendegen, L.E.; Bond, V.P.; Sondhaus, C.A.

    1998-12-31

    This report examines the origin of tissue effects that may follow from different cellular responses to low-dose irradiation, using published data. Two principal categories of cellular responses are considered. One response category relates to the probability of radiation-induced DNA damage. The other category consists of low-dose induced metabolic changes that induce mechanisms of DNA damage mitigation, which do not operate at high levels of exposure. Modeled in this way, tissue is treated as a complex adaptive system. The interaction of the various cellular responses results in a net tissue dose-effect relation that is likely to deviate from linearity in themore » low-dose region. This suggests that the LNT hypothesis should be reexamined. This paper aims at demonstrating tissue effects as an expression of cellular responses, both damaging and defensive, in relation to the energy deposited in cell mass, by use of microdosimetric concepts.« less

  8. Deficient DNA damage response and cell cycle checkpoints lead to accumulation of point mutations in human embryonic stem cells.

    PubMed

    Hyka-Nouspikel, Nevila; Desmarais, Joëlle; Gokhale, Paul J; Jones, Mark; Meuth, Mark; Andrews, Peter W; Nouspikel, Thierry

    2012-09-01

    Human embryonic stem cells (hESCs) tend to lose genomic integrity during long periods of culture in vitro and to acquire a cancer-like phenotype. In this study, we aim at understanding the contribution of point mutations to the adaptation process and at providing a mechanistic explanation for their accumulation. We observed that, due to the absence of p21/Waf1/Cip1, cultured hESCs lack proper cell cycle checkpoints and are vulnerable to the kind of DNA damage usually repaired by the highly versatile nucleotide excision repair (NER) pathway. In response to UV-induced DNA damage, the majority of hESCs succumb to apoptosis; however, a subpopulation continues to proliferate, carrying damaged DNA and accumulating point mutations with a typical UV-induced signature. The UV-resistant cells retain their proliferative capacity and potential for pluripotent differentiation and are markedly less apoptotic to subsequent UV exposure. These findings demonstrate that, due to deficient DNA damage response, the modest NER activity in hESCs is insufficient to prevent increased mutagenesis. This provides for the appearance of genetically aberrant hESCs, paving the way for further major genetic changes. Copyright © 2012 AlphaMed Press.

  9. DNA-damage-induced differentiation of leukaemic cells as an anti-cancer barrier.

    PubMed

    Santos, Margarida A; Faryabi, Robert B; Ergen, Aysegul V; Day, Amanda M; Malhowski, Amy; Canela, Andres; Onozawa, Masahiro; Lee, Ji-Eun; Callen, Elsa; Gutierrez-Martinez, Paula; Chen, Hua-Tang; Wong, Nancy; Finkel, Nadia; Deshpande, Aniruddha; Sharrow, Susan; Rossi, Derrick J; Ito, Keisuke; Ge, Kai; Aplan, Peter D; Armstrong, Scott A; Nussenzweig, André

    2014-10-02

    Self-renewal is the hallmark feature both of normal stem cells and cancer stem cells. Since the regenerative capacity of normal haematopoietic stem cells is limited by the accumulation of reactive oxygen species and DNA double-strand breaks, we speculated that DNA damage might also constrain leukaemic self-renewal and malignant haematopoiesis. Here we show that the histone methyl-transferase MLL4, a suppressor of B-cell lymphoma, is required for stem-cell activity and an aggressive form of acute myeloid leukaemia harbouring the MLL-AF9 oncogene. Deletion of MLL4 enhances myelopoiesis and myeloid differentiation of leukaemic blasts, which protects mice from death related to acute myeloid leukaemia. MLL4 exerts its function by regulating transcriptional programs associated with the antioxidant response. Addition of reactive oxygen species scavengers or ectopic expression of FOXO3 protects MLL4(-/-) MLL-AF9 cells from DNA damage and inhibits myeloid maturation. Similar to MLL4 deficiency, loss of ATM or BRCA1 sensitizes transformed cells to differentiation, suggesting that myeloid differentiation is promoted by loss of genome integrity. Indeed, we show that restriction-enzyme-induced double-strand breaks are sufficient to induce differentiation of MLL-AF9 blasts, which requires cyclin-dependent kinase inhibitor p21(Cip1) (Cdkn1a) activity. In summary, we have uncovered an unexpected tumour-promoting role of genome guardians in enforcing the oncogene-induced differentiation blockade in acute myeloid leukaemia.

  10. γδ T cells as early sensors of tissue damage and mediators of secondary neurodegeneration

    PubMed Central

    Gelderblom, Mathias; Arunachalam, Priyadharshini; Magnus, Tim

    2014-01-01

    Spontaneous or medically induced reperfusion occurs in up to 70% of patients within 24 h after cerebral ischemia. Reperfusion of ischemic brain tissue can augment the inflammatory response that causes additional injury. Recently, T cells have been shown to be an essential part of the post-ischemic tissue damage, and especially IL-17 secreting T cells have been implicated in the pathogenesis of a variety of inflammatory reactions in the brain. After stroke, it seems that the innate γδ T cells are the main IL-17 producing cells and that the γδ T cell activation constitutes an early and mainly damaging immune response in stroke. Effector mechanism of γδ T cell derived IL-17 in the ischemic brain include the induction of metalloproteinases, proinflammatory cytokines and neutrophil attracting chemokines, leading to a further amplification of the detrimental inflammatory response. In this review, we will give an overview on the concepts of γδ T cells and IL-17 in stroke pathophysiology and on their potential importance for human disease conditions. PMID:25414640

  11. A H2S-Nampt Dependent Energetic Circuit Is Critical to Survival and Cytoprotection from Damage in Cancer Cells

    PubMed Central

    Sanokawa-Akakura, Reiko; Ostrakhovitch, Elena A.; Akakura, Shin; Goodwin, Scott; Tabibzadeh, Siamak

    2014-01-01

    We recently demonstrated that cancer cells that recover from damage exhibit increased aerobic glycolysis, however, the molecular mechanism by which cancer cells survive the damage and show increased aerobic glycolysis remains unknown. Here, we demonstrate that diverse cancer cells that survive hypoxic or oxidative damage show rapid cell proliferation, and develop tolerance to damage associated with increased production of hydrogen sulfide (H2S) which drives up-regulation of nicotinamide phosphoribosyltransferase (Nampt). Consistent with existence of a H2S-Nampt energetic circuit, in damage recovered cancer cells, H2S, Nampt and ATP production exhibit a significant correlation. Moreover, the treatment of cancer cells with H2S donor, NaHS, coordinately increases Nampt and ATP levels, and protects cells from drug induced damage. Inhibition of cystathionine beta synthase (CBS) or cystathionase (CTH), enzymes which drive generation of H2S, decreases Nampt production while suppression of Nampt pathway by FK866, decreases H2S and ATP levels. Damage recovered cells isolated from tumors grown subcutaneously in athymic mice also show increased production of H2S, Nampt and ATP levels, associated with increased glycolysis and rapid proliferation. Together, these data show that upon recovery from potential lethal damage, H2S-Nampt directs energy expenditure and aerobic glycolysis in cancer cells, leads to their exponential growth, and causes a high degree of tolerance to damage. Identification of H2S-Nampt as a pathway responsible for induction of damage tolerance in cancer cells may underlie resistance to therapy and offers the opportunity to target this pathway as a means in treatment of cancer. PMID:25248148

  12. DNA Damage during G2 Phase Does Not Affect Cell Cycle Progression of the Green Alga Scenedesmus quadricauda

    PubMed Central

    Vítová, Milada; Bišová, Kateřina; Zachleder, Vilém

    2011-01-01

    DNA damage is a threat to genomic integrity in all living organisms. Plants and green algae are particularly susceptible to DNA damage especially that caused by UV light, due to their light dependency for photosynthesis. For survival of a plant, and other eukaryotic cells, it is essential for an organism to continuously check the integrity of its genetic material and, when damaged, to repair it immediately. Cells therefore utilize a DNA damage response pathway that is responsible for sensing, reacting to and repairing damaged DNA. We have studied the effect of 5-fluorodeoxyuridine, zeocin, caffeine and combinations of these on the cell cycle of the green alga Scenedesmus quadricauda. The cells delayed S phase and underwent a permanent G2 phase block if DNA metabolism was affected prior to S phase; the G2 phase block imposed by zeocin was partially abolished by caffeine. No cell cycle block was observed if the treatment with zeocin occurred in G2 phase and the cells divided normally. CDKA and CDKB kinases regulate mitosis in S. quadricauda; their kinase activities were inhibited by Wee1. CDKA, CDKB protein levels were stabilized in the presence of zeocin. In contrast, the protein level of Wee1 was unaffected by DNA perturbing treatments. Wee1 therefore does not appear to be involved in the DNA damage response in S. quadricauda. Our results imply a specific reaction to DNA damage in S. quadricauda, with no cell cycle arrest, after experiencing DNA damage during G2 phase. PMID:21603605

  13. LED-activated pheophorbide a induces cellular destruction of colon cancer cells

    NASA Astrophysics Data System (ADS)

    Xu, C. S.; Leung, A. W. N.; Liu, L.; Xia, X. S.

    2010-07-01

    Pheophorbide a (Pa) from Chinese herbal medicine Scutellaria Barbata and Silkworm Excreta shows an important promise in the photodynamic therapy on malignant tumor. The present study investigated that LED-activated Pa induced the cellular destruction of colon cancer HT-29 cells. The results showed that Pa resulted in a drug-dose dependent photocytotoxicity in the HT-29 cells, meaning the photocytotoxicity of Pa depends on the drug concentration (0 - 2 μM). We further investigated the apoptosis of the HT-29 cells 18 hours after photosensitization of Pa using a confocal laser scanning microscopy with Hoechst 33258 staining. These data demonstrated that LED-activated Pa could significantly induce the cellular destruction of the HT-29 cells.

  14. TRIM39 regulates cell cycle progression and DNA damage responses via stabilizing p21

    PubMed Central

    Zhang, Lei; Mei, Yang; Fu, Nai-yang; Guan, Li; Xie, Wei; Liu, Hui-hui; Yu, Chun-dong; Yin, Zhenyu; Yu, Victor C.; You, Han

    2012-01-01

    The biological function of Tripartite Motif 39 (TRIM39) remains largely unknown. In this study, we report that TRIM39 regulates the steady-state levels of p21 and is a pivotal determinant of cell fate. Ablation of TRIM39 leads to destabilization of p21 and increased G1/S transition in unperturbed cells. Furthermore, DNA damage-induced p21 accumulation is completely abolished in cells with depleted TRIM39. As a result, silencing of TRIM39 abrogates the G2 checkpoint induced by genotoxic stress, leading to increased mitotic entry and, ultimately, apoptosis. Importantly, we show p21 is a crucial downstream effector of TRIM39 mediating G1/S transition and DNA damage-induced G2 arrest. Mechanistically, TRIM39 interacts with p21, which subsequently prevents Cdt2 from binding to p21, therefore blocking ubiquitylation and proteasomal degradation of p21 mediated by CRL4Cdt2 E3 ligase. Strikingly, we found a significant correlation between p21 abundance and TRIM39 expression levels in human hepatocellular carcinoma samples. Our findings identify a causal role for TRIM39 in regulating cell cycle progression and the balance between cytostasis and apoptosis after DNA damage via stabilizing p21. PMID:23213251

  15. Evaluation of Cassia tora Linn. against Oxidative Stress-induced DNA and Cell Membrane Damage

    PubMed Central

    Kumar, R Sunil; Narasingappa, Ramesh Balenahalli; Joshi, Chandrashekar G; Girish, Talakatta K; Prasada Rao, Ummiti JS; Danagoudar, Ananda

    2017-01-01

    Objective: The present study aims to evaluate antioxidants and protective role of Cassia tora Linn. against oxidative stress-induced DNA and cell membrane damage. Materials and Methods: The total and profiles of flavonoids were identified and quantified through reversed-phase high-performance liquid chromatography. In vitro antioxidant activity was determined using standard antioxidant assays. The protective role of C. tora extracts against oxidative stress-induced DNA and cell membrane damage was examined by electrophoretic and scanning electron microscopic studies, respectively. Results: The total flavonoid content of CtEA was 106.8 ± 2.8 mg/g d.w.QE, CtME was 72.4 ± 1.12 mg/g d.w.QE, and CtWE was 30.4 ± 0.8 mg/g d.w.QE. The concentration of flavonoids present in CtEA in decreasing order: quercetin >kaempferol >epicatechin; in CtME: quercetin >rutin >kaempferol; whereas, in CtWE: quercetin >rutin >kaempferol. The CtEA inhibited free radical-induced red blood cell hemolysis and cell membrane morphology better than CtME as confirmed by a scanning electron micrograph. CtEA also showed better protection than CtME and CtWE against free radical-induced DNA damage as confirmed by electrophoresis. Conclusion: C. tora contains flavonoids and inhibits oxidative stress and can be used for many health benefits and pharmacotherapy. PMID:28584491

  16. DNA damage in oral cancer and normal cells induced by nitrogen atmospheric pressure plasma jets

    NASA Astrophysics Data System (ADS)

    Han, Xu; Kapaldo, James; Liu, Yueying; Stack, M. Sharon; Ptasinska, Sylwia

    2015-09-01

    Nitrogen atmospheric pressure plasma jets (APPJs) have been shown to effectively induce DNA double strand breaks in SCC25 oral cancer cells. The APPJ source constructed in our laboratory operates based on dielectric barrier discharge. It consists of two copper electrodes alternatively wrapping around a fused silica tube with nitrogen as a feed gas. It is generally more challenging to ignite plasma in N2 atmosphere than in noble gases. However, N2 provides additional advantages such as lower costs compared to noble gases, thus this design can be beneficial for the future long-term clinical use. To compare the effects of plasma on cancer cells (SCC25) and normal cells (OKF), the cells from both types were treated at the same experimental condition for various treatment times. The effective area with different damage levels after the treatment was visualized as 3D maps. The delayed damage effects were also explored by varying the incubation times after the treatment. All of these studies are critical for a better understanding of the damage responses of cellular systems exposed to the plasma radiation, thus are useful for the development of the advanced plasma cancer therapy. The research described herein was supported by the Division of Chemical Sciences, Geosciences and Biosciences, Basic Energy Sciences, Office of Science, United States Department of Energy through Grant No. DE-FC02-04ER15533.

  17. Single-molecule live-cell imaging of bacterial DNA repair and damage tolerance.

    PubMed

    Ghodke, Harshad; Ho, Han; van Oijen, Antoine M

    2018-02-19

    Genomic DNA is constantly under threat from intracellular and environmental factors that damage its chemical structure. Uncorrected DNA damage may impede cellular propagation or even result in cell death, making it critical to restore genomic integrity. Decades of research have revealed a wide range of mechanisms through which repair factors recognize damage and co-ordinate repair processes. In recent years, single-molecule live-cell imaging methods have further enriched our understanding of how repair factors operate in the crowded intracellular environment. The ability to follow individual biochemical events, as they occur in live cells, makes single-molecule techniques tremendously powerful to uncover the spatial organization and temporal regulation of repair factors during DNA-repair reactions. In this review, we will cover practical aspects of single-molecule live-cell imaging and highlight recent advances accomplished by the application of these experimental approaches to the study of DNA-repair processes in prokaryotes. © 2018 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.

  18. Lineage-Specific Genes Are Prominent DNA Damage Hotspots during Leukemic Transformation of B Cell Precursors.

    PubMed

    Boulianne, Bryant; Robinson, Mark E; May, Philippa C; Castellano, Leandro; Blighe, Kevin; Thomas, Jennifer; Reid, Alistair; Müschen, Markus; Apperley, Jane F; Stebbing, Justin; Feldhahn, Niklas

    2017-02-14

    In human leukemia, lineage-specific genes represent predominant targets of deletion, with lymphoid-specific genes frequently affected in lymphoid leukemia and myeloid-specific genes in myeloid leukemia. To investigate the basis of lineage-specific alterations, we analyzed global DNA damage in primary B cell precursors expressing leukemia-inducing oncogenes by ChIP-seq. We identified more than 1,000 sensitive regions, of which B lineage-specific genes constitute the most prominent targets. Identified hotspots at B lineage genes relate to DNA-DSBs, affect genes that harbor genomic lesions in human leukemia, and associate with ectopic deletion in successfully transformed cells. Furthermore, we show that most identified regions overlap with gene bodies of highly expressed genes and that induction of a myeloid lineage phenotype in transformed B cell precursors promotes de novo DNA damage at myeloid loci. Hence, we demonstrate that lineage-specific transcription predisposes lineage-specific genes in transformed B cell precursors to DNA damage, which is likely to promote the frequent alteration of lineage-specific genes in human leukemia. Copyright © 2017 The Author(s). Published by Elsevier Inc. All rights reserved.

  19. HIPK2 knock-down compromises tumor cell efficiency to repair damaged DNA.

    PubMed

    Nardinocchi, Lavinia; Puca, Rosa; Sacchi, Ada; D'Orazi, Gabriella

    2007-09-14

    Homeodomain Interacting Protein Kinase-2 (HIPK2) is a protein with many functions and a modulator of p53 oncosuppressor functions. TP53 is the "guardian of the genome" thus, is the most critical tumor suppressor gene product that inhibits malignant transformation. P53R2 gene is directly induced by p53 in response to DNA damage and is involved in the p53 checkpoint for repairing damaged DNA to block genome instability. Here we wanted to explore the involvement of HIPK2 in damaged-DNA repair by regulating p53-induced p53R2 gene. We show that, induction of p53R2 expression, p53 recruitment onto p53R2 promoter, and its transcriptional activation was strongly impaired by HIPK2 knock-down, in response to drug. The failure of p53-induced p53R2 activation markedly compromised damaged-DNA repair efficiency. Finally, overexpression of exogenous p53 overcame the inability of endogenous p53 to activate p53R2-luc promoter in HIPK2 depleted cells. These data suggest that HIPK2 is involved in damaged-DNA repair taking part in restraining tumor progression, at least in part depending on p53 regulation.

  20. [Macrocalin A induces apoptosis of multiple myeloma U266 cells through inhibiting the proteasome].

    PubMed

    Lu, Ling-Na; Feng, Li-Qian; Lu, Ya-Ping; Xia, Jun; Qiu, Lian Nu; Shi, Hao; Wang, Wei-Zhong; Zhou, Yong-Lie

    2013-12-01

    This study was purposed to investigate the inhibitory effect of macrocalin A (MA) on proteasome of multiple myeloma U266 cells in vitro and molecular mechanism of MA-inducing apoptosis. U266 cells in vitro were incubated with different concentrations (2, 4, 8 µg/mL) of MA, the Hochest staining and Annexin-V/PI double staining were used to detect the apoptosis of U266 cells. The expressions of protein β1, β1i, β2, β2i, β5, β5i, ubiquitous, 19S subunit S6', and BAD,BCL-2, FAS, FAS-L,MAPK, PARP, Pro-caspase 3, cleaved-caspase 3 were detected by Western blot technique. The results showed that along with time prolonging and dose increasing of MA, the small and compact fluorescent particles were observed in cytoplasm and nucleus of U266 cells stained with Hoechst 33258, the Annexin V(+)/PI(-) cells and the total apoptosis cells (Annexin V(+)/PI(-) and Annexin V(+)/PI(+)) increased. MA could elevate the ubiquitylation level in U266 cells, suppress the expression of β1i,β2, β5i and 19S subunit S6', meanwhile the expression of BCJ-2, MAPK, PARP and pro-caspase 3 were down-regulated along with increasing of drug concentrations, but the expressions of BAD, FAS, FAS-L cleaved-caspase 3 were enhanced. It is concluded that MA can inhibit the effect of proteasome, and the mitochondrial pathway and death receptor pathway may play important roles in apoptosis of U266 cells induced by MA.

  1. Levistolide A Induces Apoptosis via ROS-Mediated ER Stress Pathway in Colon Cancer Cells.

    PubMed

    Yang, Yingjuan; Zhang, Yanhua; Wang, Lan; Lee, Shaochin

    2017-01-01

    Colorectal cancer (CRC) is one of the leading causes of cancer-related death worldwide. Levistolide A (LA), a natural compound isolated from the traditional Chinese herb Ligusticum chuanxiong Hort, is used for treating cancer. In this study, we investigated the anticancer effect of LA in HCT116 and its isogenic p53-/- colon cancer cells, as well as the underlying mechanisms. MTT assay was used to evaluate the effect of LA on the viability of cancer cells. Apoptosis and reactive oxygen species (ROS) production by the cells were determined by flow cytometry. Protein expression was detected by western blotting. The results showed that LA inhibited viability and caused apoptosis of both wild-type and p53-/- HCT116 cells. LA was able to trigger production of ROS and endoplasmic reticulum (ER) stress. Inhibition of ROS using N-acetylcysteine abrogated LA-induced ER stress and apoptosis, as well as the reduction in cancer cell viability. Our results indicate that LA causes apoptosis of colon cancer cells via ROS-mediated ER stress pathway. It will be interesting to develop the natural compound for chemotherapy of cancers such as CRC. © 2017 The Author(s). Published by S. Karger AG, Basel.

  2. Dental pulp stem cells promote regeneration of damaged neuron cells on the cellular model of Alzheimer's disease.

    PubMed

    Wang, Feixiang; Jia, Yali; Liu, Jiajing; Zhai, Jinglei; Cao, Ning; Yue, Wen; He, Huixia; Pei, Xuetao

    2017-06-01

    Alzheimer's disease (AD) is an incurable neurodegenerative disease and many types of stem cells have been used in AD therapy with some favorable effects. In this study, we investigated the potential therapeutical effects of human dental pulp stem cells (hDPSCs) on AD cellular model which established by okadaic acid (OA)-induced damage to human neuroblastoma cell line, SH-SY5Y, in vitro for 24 h. After confirmed the AD cellular model, the cells were co-culture with hDPSCs by transwell co-culture system till 24 h for treatment. Then the cytomorphology of the hDPSCs-treated cells were found to restore gradually with re-elongation of retracted dendrites. Meanwhile, Cell Counting Kit-8 assay and Hoechst 33258 staining showed that hDPSCs caused significant increase in the viability and decrease in apoptosis of the model cells, respectively. Observation of DiI labeling also exhibited the prolongation dendrites in hDPSCs-treated cells which were obviously different from the retraction dendrites in AD model cells. Furthermore, specific staining of α-tubulin and F-actin demonstrated that the hDPSCs-treated cells had the morphology of restored neurons, with elongated dendrites, densely arranged microfilaments, and thickened microtubular fibrils. In addition, results from western blotting revealed that phosphorylation at Ser 396 of Tau protein was significantly suppressed by adding of hDPSCs. These results indicate that hDPSCs may promote regeneration of damaged neuron cells in vitro model of AD and may serve as a useful cell source for treatment of AD. © 2017 International Federation for Cell Biology.

  3. Cytogenetic damage, oncogenic transformation and p53 induction in human epithelial cells in response to irradiation

    NASA Astrophysics Data System (ADS)

    Armitage, Mark

    Ionizing radiation can have several different effects on cells, some are almost instantaneous such as the generation of DNA damage, other cellular responses take a matter of minutes or hours - DNA repair protein induction/activation, and others may take months or even years to be manifested - carcinogenesis. Human epithelial cell lines derived from both normal, non-neoplastic tissues and from a malignant source were cultured in order to examine several effects of ionizing radiation on such cell types. Cells not from a malignant source were previously immortalized by viral infection or by transfection with viral sequences. Simian virus 40 immortalised uroepithelial cells (SV-HUC) were found to be approximately a factor of two fold more radioresistant than cells of malignant origin (T24) in terms of unrepaired clastogenic damage i.e. assessment of micronuclei levels following irradiation. SV-HUC lines unlike T24 cells are non-tumourigenic when inoculated into nude athymic mice. SV-HUC lines proved very resistant to full oncogenic transformation using radiation and chemical carcinogens. However, morphological alterations and decreased anchorage dependant growth was observed in post carcinogen treated cells after appropriate cell culture conditions were utilized. The progression from this phenotype to a fully tumourigenic one was not recorded in this study. The ability of ionizing radiation to induce increased levels of the nuclear phosphoprotein p53 was also assessed using several different cell lines. SV- HUC and T24 cell lines failed to exhibit any increased p53 stabilization following irradiation. One cell line, a human papilloma virus transformed line (HPV) did show an approximate two fold increase of the wild type p53 protein after treatment with radiation. Only the cell line HPV showed any cell cycle delay, resulting in accumulation of cells in the G2/M compartment in post irradiation cell cycle analysis. The status of p53 was also assessed i.e. wild type or

  4. Abnormal cytokinesis after X-irradiation in tumor cells that override the G2 DNA damage checkpoint.

    PubMed

    Huang, Haomin; Fletcher, Lynda; Beeharry, Neil; Daniel, Rene; Kao, Gary; Yen, Tim J; Muschel, Ruth J

    2008-05-15

    X-irradiation-induced DNA damage perturbs the G(1), S, and G(2) phases of the cell cycle. The behavior of cells after they have experienced a DNA damage checkpoint delay is poorly characterized. We therefore examined the fates of irradiated tumor cells that have overcome a prolonged G(2) checkpoint delay. Most irradiated cells progressed through mitosis without significant delay, but failed to complete cytokinesis as they remained tethered to each other at the midbody. We observed that the movement of centrioles at the time of cytokinesis was impaired in the irradiated, bridged cells. We attribute the perturbation of centriole dynamics to the presence of chromatin bridges that spanned the daughter cells. The bridged cells exhibited different fates that included death, fusion that formed multinucleated cells, or another round of mitosis with no noticeable cell cycle delays. The presence of gammaH2AX foci in the bridge as well as in the separated nuclei indicated that cells were proliferating despite the presence of DNA damage. It seems that DNA damage checkpoints were not reactivated in cells that overrode a prolonged G(2) delay. Cells deficient in ATM, H2AX, XRCC3, or ligase 4 exhibited a higher frequency of radiation-induced bridges than controls, suggesting that the DNA bridges resulted from inadequate DNA repair. These data show a previously unappreciated cytologic hallmark of DNA damage in dividing cells. Chromatin bridges that interfere with cytokinesis are likely to contribute to the replication failure and clonogenic death of cells exposed to irradiation.

  5. SIRT6 protects human endothelial cells from DNA damage, telomere dysfunction, and senescence.

    PubMed

    Cardus, Anna; Uryga, Anna K; Walters, Gareth; Erusalimsky, Jorge D

    2013-03-01

    Although endothelial cell senescence is known to play an important role in the development of cardiovascular pathologies, mechanisms that attenuate this process have not been extensively investigated. The aim of this study was to investigate whether SIRT6, a member of the sirtuin family of NAD(+)-dependent protein deacetylases/ADP-ribosyltransferases, protects endothelial cells from premature senescence and dysfunction, and if so which is its mode of action. mRNA expression analysis demonstrated comparable levels of SIRT1 and SIRT6 transcripts in endothelial cells derived from different vascular beds and significantly higher levels of SIRT6 in these cells relative to those in haematopoietic progenitor cells. SIRT6 depletion by RNA interference in human umbilical vein endothelial cells (HUVEC) and aortic endothelial cells reduced cell proliferation, increased the fraction of senescence-associated-β-galactosidase-positive cells, and diminished the ability of the cells to form tubule networks on Matrigel. Further examination of SIRT6-depleted HUVEC demonstrated higher intercellular-adhesion molecule-1 (ICAM-1) and plasminogen-activator inhibitor-1 mRNA, lower levels of endothelial nitric oxide synthase mRNA and protein, higher ICAM-1 surface expression, and up-regulation of p21. Fluorescence microscopy of SIRT6-depleted HUVEC stained with anti-phospho-histone H2A.X and anti-telomere-repeat-binding-factor-1 antibodies showed evidence of increased nuclear DNA damage and the formation of telomere dysfunction-induced foci. This work demonstrates that the presence of SIRT6 in endothelial cells confers protection from telomere and genomic DNA damage, thus preventing a decrease in replicative capacity and the onset of premature senescence. These findings suggest that SIRT6 may be important to maintain endothelial homeostatic functions and delay vascular ageing.

  6. Intraretinal Transplantation for Rod-Cell Replacement in Light-Damaged Retinas

    PubMed Central

    del Cerro, Manuel; Notter, Mary F. D.; del Cerro, Coca; Wiegand, Stanley J.; Grover, Donald A.; Lazar, Eliot

    1989-01-01

    Blindness from retinal disease is often the consequence of extensive damage to the photoreceptor cell population, while other cell types which form the neural retina are relatively spared. In this setting, transplantation of photoreceptor cells could offer hope for the restoration of some degree of visual function. We testd the feasibility of this approach by transplanting immature retinal cells into the eyes of adult rats affected by late stage phototoxic retinopathy, which are almost totally devoid of photoreceptor cells. Dissociated neuroretinal cells from newborn rats were injected into the hosts' retinas. These cells were labelled with the fluorescent tracer Fast-blue for identification within the host eye. Survival time ranged from 3 to 100 post-transplantation days. Fundus examination of light-irradiated eyes showed pallor caused by a considerable reduction of the retino-choroidal vascular bed after light irradiation. Histologically the hosts exhibited decimation of the elements forming the outer layers.throughout the entire retina. As visualized by light and electron microscopic procedures, we report the differentiation of clusters of transplanted photoreceptor cells, and the integration of these cells within the adjacent areas of the host retina. Fluorescence microscopy showed these clusters to be formed by fluorescently labelled cells developing in intimate contact with the unlabelled host retina. Electron microscopically it was possible to determine that these photoreceptors had established synaptic contacts. These observations indicate that successful transplantation of immature retinal cells is feasible into adult eyes that have suffered extensive retino-choroidal damage. These findings also support the concept that retinal transplantation is a procedure which may open new avenues into the study of retinal repair. PMID:2519517

  7. UVB damage onset and progression 24 h post exposure in human-derived skin cells.

    PubMed

    Khalil, Christian; Shebaby, Wassim

    2017-01-01

    The focus of this research was on UVB radiation (280-320 nm) responsible for cellular changes in skin of acute and chronically exposed individuals. This study investigated the acute cellular damages triggered by UVB exposure of cultured human fibroblasts and keratinocyte cells immediately and 24 h post exposure in order to understand damage onset and progression. The study evaluated a number of cellular parameters including mitochondria, lysosomes, cell membrane, DNA damages as well as pro and anti-apoptotic protein expression levels. Cellular organelle damages were assessed by a battery of in vitro toxicological assays using MTS and Neutral red cytotoxicity assays. Cell membrane damages were also assessed by measuring lactate dehydrogenase (LDH) enzyme leakage from UVB exposed cells. Lastly DNA damages was assessed using the comet assay while protein expression was evaluated using Western Blot. In this study we reported in all our assay systems (MTS, NR and LDH) that cellular damages were UVB dose dependent with damages amplified 24 h post exposure. Our results also indicated that incubation of exposed cells for a period of 24 h increased the sensitivity of the assay systems used. The increased sensitivity in detecting early cytotoxic damages was manifested though organelle damage measurement at very low doses which were not manifested immediately post exposure. The data also indicated that HaCaT cells were most sensitive in detecting UVB triggered damages immediately and 24 h post exposure using the MTS assay. We also established upregulation and downregulation of various apoptotic proteins at various time points post exposure. The presented data clearly indicated the need for a comprehensive assessment of UVB damages 4 and 24 h post exposure due to the different assay sensitivities in addition to various signaling mechanisms activated at different time points post exposure.

  8. High molecular weight hyaluronan decreases oxidative DNA damage induced by EDTA in human corneal epithelial cells

    PubMed Central

    Ye, J; Wu, H; Wu, Y; Wang, C; Zhang, H; Shi, X; Yang, J

    2012-01-01

    Purpose To investigate the toxic effects of ethylenediaminetetraacetic acid disodium salt (EDTA), a corneal penetration enhancer in topical ophthalmic formulations, on DNA in human corneal epithelial cells (HCEs), and to investigate whether the effect induced by EDTA can be inhibited by high molecular weight hyaluronan (HA). Methods Cells were exposed to EDTA in concentrations ranging from 0.00001 to 0.01% for 60 min, or 30 min high molecular weight HA pretreatment followed by EDTA treatment. The cell viability was measured by the MTT test. Cell apoptosis was determined with annexin V staining by flow cytometry. The DNA single- and double-strand breaks of HCEs were examined by alkaline comet assay and by immunofluorescence microscope detection of the phosphorylated form of histone variant H2AX (γH2AX) foci, respectively. Reactive oxygen species (ROS) production was assessed by the fluorescent probe, 2′, 7′-dichlorodihydrofluorescein diacetate. Results EDTA exhibited no adverse effect on cell viability and did not induce cell apoptosis in human corneal epithelial cells at concentrations lower than 0.01%. However, a significant increase of DNA single- and double-strand breaks was observed in a dose-dependent manner with all the concentrations of EDTA tested in HCEs. In addition, EDTA treatment led to elevated ROS generation. Moreover, 30 min preincubation with high molecular weight HA significantly decreased EDTA-induced ROS generation and DNA damage. Conclusions EDTA could induce DNA damage in HCEs, probably through oxidative stress. Furthermore, high molecular weight HA was an effective protective agent that had antioxidant properties and decreased DNA damage induced by EDTA. PMID:22595911

  9. All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis

    PubMed Central

    Sawada, Osamu; Perusek, Lindsay; Kohno, Hideo; Howell, Scott J.; Maeda, Akiko; Matsuyama, Shigemi; Maeda, Tadao

    2014-01-01

    The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated X protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661W cells, cultured neural retinas and a retinal degeneration model, Abca4−/−Rdh8−/− mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser 46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4−/−Rdh8−/− mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis. PMID:24726920

  10. All-trans-retinal induces Bax activation via DNA damage to mediate retinal cell apoptosis.

    PubMed

    Sawada, Osamu; Perusek, Lindsay; Kohno, Hideo; Howell, Scott J; Maeda, Akiko; Matsuyama, Shigemi; Maeda, Tadao

    2014-06-01

    The current study investigates the cellular events which trigger activation of proapoptotic Bcl-2-associated × protein (Bax) in retinal cell death induced by all-trans-retinal (atRAL). Cellular events which activate Bax, such as DNA damage by oxidative stress and phosphorylation of p53, were evaluated by immunochemical and biochemical methods using ARPE-19 cells, 661 W cells, cultured neural retinas and a retinal degeneration model, Abca4(-/-)Rdh8(-/-) mice. atRAL-induced Bax activation in cultured neural retinas was examined by pharmacological and genetic methods. Other Bax-related cellular events were also evaluated by pharmacological and biochemical methods. Production of 8-OHdG, a DNA damage indicator, and the phosphorylation of p53 at Ser46 were detected prior to Bax activation in ARPE-19 cells incubated with atRAL. Light exposure to Abca4(-/-)Rdh8(-/-) mice also caused the above mentioned events in conditions of short term intense light exposure and regular room lighting conditions. Incubation with Bax inhibiting peptide and deletion of the Bax gene partially protected retinal cells from atRAL toxicity in cultured neural retina. Necrosis was demonstrated not to be the main pathway in atRAL mediated cell death. Bcl-2-interacting mediator and Bcl-2 expression levels were not altered by atRAL in vitro. atRAL-induced oxidative stress results in DNA damage leading to the activation of Bax by phosphorylated p53. This cascade is closely associated with an apoptotic cell death mechanism rather than necrosis. Copyright © 2014 Elsevier Ltd. All rights reserved.

  11. DNA damage response (DDR) via NKX3.1 expression in prostate cells.

    PubMed

    Erbaykent-Tepedelen, Burcu; Karamil, Selda; Gonen-Korkmaz, Ceren; Korkmaz, Kemal S

    2014-05-01

    It has been reported that NKX3.1 an androgen-regulated homeobox gene restricted to prostate and testicular tissues, encodes a homeobox protein, which transcriptionally regulates oxidative damage responses and enhances topoisomerase I re-ligation by a direct interaction with the ATM protein in prostate cells. In this study, we aimed to investigate the role of NKX3.1 in DNA double-strand break (DSB) repair. We demonstrate that the DNA damage induced by CPT-11 (irinotecan, a topo I inhibitor), doxorubicin (a topo II inhibitor), and H2O2 (a mediator of oxidative damage), but not by etoposide (another topo II inhibitor), is negatively influenced by NKX3.1 expression. We also examined γH2AX((S139)) foci formation and observed that the overexpression of NKX3.1 resulted a remarkable decrease in the formation of γH2AX((S139)) foci. Intriguingly, we observed in NKX3.1 silencing studies that the depletion of NKX3.1 correlated with a significant decrease in the levels of p-ATM((S1981)) and γH2AX((S139)). The data imply that the DNA damage response (DDR) can be altered, perhaps via a decrease in the topoisomerase I re-ligation function; this is consistent with the physical association of NKX3.1 with DDR mediators upon treatment of both PC-3 and LNCaP cells with CPT-11. Furthermore, the depletion of NKX3.1 resulted in a G1/S progression via the facilitation of an increase in E2F stabilization concurrent with the suppressed DDR. Thus, the topoisomerase I inhibitor-mediated DNA damage enhanced the physical association of NKX3.1 with γH2AX((S139)) on the chromatin in LNCaP cells, whereas NKX3.1 in the soluble fraction was associated with p-ATM((S1981)) and RAD50 in these cells. Overall, the data suggest that androgens and NKX3.1 expression regulate the progression of the cell cycle and concurrently activate the DDR. Therefore, androgen withdrawal may augment the development of an error-prone phenotype and, subsequently, the loss of DNA damage control during prostate cancer

  12. Effects of melatonin and its receptor antagonist on retinal pigment epithelial cells against hydrogen peroxide damage

    PubMed Central

    Rosen, Richard B.; Hu, Dan-Ning; Chen, Min; McCormick, Steven A.; Walsh, Joseph

    2012-01-01

    Purpose Recently, we reported finding that circulating melatonin levels in age-related macular degeneration patients were significantly lower than those in age-matched controls. The purpose of this study was to investigate the hypothesis that melatonin deficiency may play a role in the oxidative damage of the retinal pigment epithelium (RPE) by testing the protective effect of melatonin and its receptor antagonist on RPE cells exposed to H2O2 damage. Methods Cultured human RPE cells were subjected to oxidative stress induced by 0.5 mM H2O2. Cell viability was measured using the microculture tetrazoline test (MTT) assay. Cells were pretreated with or without melatonin for 24 h. Luzindole (50 μM), a melatonin membrane-receptor antagonist, was added to the culture 1 h before melatonin to distinguish direct antioxidant effects from indirect receptor-dependent effects. All tests were performed in triplicate. Results H2O2 at 0.5 mM decreased cell viability to 20% of control levels. Melatonin showed dose-dependent protective effects on RPE cells against H2O2. Cell viability of RPE cells pretreated with 10−10, 10−8, 10−6, and 10−4 M melatonin for 24 h was 130%, 160%, 187%, and 230% of cells treated with H2O2 alone (all p<0.05). Using cells cultured without H2O2 as the control, cell viability of cells treated with H2O2 after pretreatment with 10−10-10−4 M melatonin was still significantly lower than that of the controls, suggesting that melatonin significantly decreased but did not completely abolish the in vitro cytotoxic effects of H2O2. Luzindole completely blocked melatonin’s protective effects at low concentrations of melatonin (10−10-10−8 M) but not at high concentrations (10−6-10−4 M). Conclusions Melatonin has a partial protective effect on RPE cells against H2O2 damage across a wide range of concentrations (10−10-10−4 M). This protective effect occurs through the activation of melatonin membrane receptors at low concentrations (10−10

  13. Carbosilane dendrimers inhibit α-synuclein fibrillation and prevent cells from rotenone-induced damage.

    PubMed

    Milowska, Katarzyna; Szwed, Aleksandra; Mutrynowska, Marta; Gomez-Ramirez, Rafael; de la Mata, Francisco Javier; Gabryelak, Teresa; Bryszewska, Maria

    2015-04-30

    This study investigates the role of carbosilane dendrimers in fibrillation of α-synuclein and prevention of the mouse hippocampal cell (mHippoE-18) from rotenone-induced damage. Examining the interaction between carbosilane dendrimers and α-synuclein, we found that the dendrimers inhibit fibril formation. We also investigated cell viability, the production of reactive oxygen species (ROS), and mitochondrial membrane potential. mHippoE-18 cells were preincubated with carbosilane dendrimers before rotenone was added. All the dendrimers possess potential protection activity. Preincubation with dendrimers contributed to: increased viability, higher mitochondrial membrane potential, and reduced ROS level in cells. The probable mechanism of cell protection lies in the ability of dendrimers to capture rotenone by encapsulating or binding to its surface groups. The fact that dendrimers have prevention potential is important in the search for new pharmacological strategies against neurodegenerative disorders. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. Cellular track model of biological damage to mammalian cell cultures from galactic cosmic rays

    NASA Technical Reports Server (NTRS)

    Cucinotta, Francis A.; Katz, Robert; Wilson, John W.; Townsend, Lawrence W.; Nealy, John E.; Shinn, Judy L.

    1991-01-01

    The assessment of biological damage from the galactic cosmic rays (GCR) is a current interest for exploratory class space missions where the highly ionizing, high-energy, high-charge ions (HZE) particles are the major concern. The relative biological effectiveness (RBE) values determined by ground-based experiments with HZE particles are well described by a parametric track theory of cell inactivation. Using the track model and a deterministic GCR transport code, the biological damage to mammalian cell cultures is considered for 1 year in free space at solar minimum for typical spacecraft shielding. Included are the effects of projectile and target fragmentation. The RBE values for the GCR spectrum which are fluence-dependent in the track model are found to be more severe than the quality factors identified by the International Commission on Radiological Protection publication 26 and seem to obey a simple scaling law with the duration period in free space.

  15. Titanium dioxide induced cell damage: a proposed role of the carboxyl radical.

    PubMed

    Dodd, Nicholas J F; Jha, Awadhesh N

    2009-01-15

    Titanium dioxide (TiO(2)) nanoparticles have been shown to be genotoxic to cells exposed to ultraviolet A (UVA) radiation. Using the technique of electron spin resonance (ESR) spin trapping, we have confirmed that the primary damaging species produced on irradiation of TiO(2) nanoparticles is the hydroxyl (OH) radical. We have applied this technique to TiO(2)-treated fish and mammalian cells under in vitro conditions and observed the additional formation of carboxyl radical anions (CO(2)(-)) and superoxide radical anions (O(2)(-)). This novel finding suggests a hitherto unreported pathway for damage, involving primary generation of OH radicals in the cytoplasm, which react to give CO(2)(-) radicals. The latter may then react with cellular oxygen to form O(2)(-) and genotoxic hydrogen peroxide (H(2)O(2)).

  16. 2-Nitroanisole-induced oxidative DNA damage in Salmonella typhimurium and in rat urinary bladder cells.

    PubMed

    Wada, Kunio; Katoh, Yoshitaka; Ohnuma-Koyama, Aya; Takahashi, Naofumi; Yamada, Masami; Matsumoto, Kyomu

    2017-04-01

    2-Nitroanisole (2-NA) is used in the manufacturing of azo dyes and causes cancer, mainly in the urinary bladder. Previous in vivo genotoxic data seems to be insufficient to explain the mechanism through which 2-NA induces carcinogenesis, and several bladder carcinogens were reported to induce oxidative DNA damage. Thus, we examined the potential induction of oxidative DNA damage by 2-NA using bacterial strain YG3008, a mutM ST -deficient derivative of strain TA100. Consequently, strain YG3008, when compared with strain TA100, was found to be more sensitive to 2-NA, indicating oxidative DNA damage in bacterial cells. For further investigation, we performed the comet assay using the urinary bladder and liver of rats, with and without human 8-oxoguanine DNA-glycosylase 1 (hOGG1), to confirm the potential of 2-NA for inducing oxidative DNA damage. Simultaneously, we conducted a micronucleus test using bone marrow from rats to assess the genotoxicity of 2-NA in vivo. 2-NA was administered orally to male Fischer 344 rats for 3 consecutive days. The rats were divided into 6 treatment groups: 3 groups treated with 2-NA at doses of 125, 250, and 500mg/kg; a group treated with the combination of 2-NA and glutathione-SH (GSH); a negative control group; and a positive control group. The comet assay without hOGG1 detected no DNA damage in the liver or urinary bladder, and the micronucleus test did not show clastogenic effects in bone marrow cells. However, the comet assay with hOGG1 was positive in the urinary bladder samples, indicating the induction of oxidative DNA damage in the urinary bladder for the group treated with 2-NA at 500mg/kg. Moreover, an antioxidant of GSH significantly reduced oxidative DNA damage caused by 2-NA. These results indicate that oxidative DNA damage is a possible mode of action for carcinogenesis in the urinary bladder of rats treated with 2-NA. Copyright © 2017 Elsevier B.V. All rights reserved.

  17. Hydrophobicity of whey protein hydrolysates enhances the protective effect against oxidative damage on PC 12 cells.

    PubMed

    Zhang, Qiu-Xiang; Jin, Man-Man; Zhang, Li; Yu, Hui-Xin; Sun, Zhen; Lu, Rong-Rong

    2015-02-01

    The relationship between hydrophobicity and the protective effect of whey protein hydrolysates (WPHs) against oxidative stress was studied. Whey protein was first hydrolysed by pepsin and trypsin to obtain WPHs. After absorbed by macroporous adsorption resin DA201-C, three fractions named as M20, M40, and M60 were eluted by various concentrations of ethanol. The hydrophobicity showed a trend of increase from M20 to M60. Antioxidant ability test in vitro indicated that all the three components of WPHs displayed reasonably good antioxidant ability. Moreover, with the increase of hydrophobicity, antioxidant ability of WPHs improved significantly. Then rat pheochromocytoma line 12 (PC12) cells oxidative model was built to evaluate the suppression of oxidative stress of three components on PC12 cells induced by H2O2. Morphological alterations, cell viability, apoptosis rate, and intracellular antioxidase system tests all indicated that WPHs exert significant protection on PC cells against H2O2-induced damage. Among them, M60 had the highest protective effect by increasing 19·3% cell survival and reducing 28·6% cell apoptosis. These results suggested hydrophobicity of WPHs was contributing to the antioxidant ability and the protective effect against oxidative damage.

  18. Induction of phase 2 genes by sulforaphane protects retinal pigment epithelial cells against photooxidative damage

    PubMed Central

    Gao, Xiangqun; Talalay, Paul

    2004-01-01

    The retinal pigment epithelial cell (RPE cell) layer protects the photoreceptors of the retina against oxidative stress. The decline of this capacity is believed to be a major factor in the impairment of vision in age-related macular degeneration. Exposure of human adult RPE cells to UV light at predominantly 320–400 nm (UVA light) in the presence of all-trans-retinaldehyde results in photooxidative cytotoxicity. Significant protection of RPE cells was obtained by prior treatment with phase 2 gene inducers, such as the isothiocyanate sulforaphane or a bis-2-hydroxybenzylideneacetone Michael reaction acceptor. The degree of protection was correlated with the potencies of these inducers in elevating cytoprotective glutathione levels and activities of NAD(P)H:quinone oxidoreductase. In embryonic fibroblasts derived from mice in which the genes for the transcription factor Nrf2, the repressor Keap1, or both Nrf2 and Keap1 were disrupted, the magnitude of resistance to photooxidative damage paralleled the basal levels of glutathione and NAD(P)H:quinone oxidoreductase in each cell type. Demonstration of protection of RPE cells against photooxidative damage by induction of phase 2 proteins may shed light on the role of oxidative injury in ocular disease. Moreover, the finding that dietary inducers provide indirect antioxidant protection suggests novel strategies for preventing chronic degenerative diseases, such as age-related macular degeneration. PMID:15229324

  19. Deficiency in DNA damage response, a new characteristic of cells infected with latent HIV-1.

    PubMed

    Piekna-Przybylska, Dorota; Sharma, Gaurav; Maggirwar, Sanjay B; Bambara, Robert A

    2017-05-19

    Viruses can interact with host cell molecules responsible for the recognition and repair of DNA lesions, resulting in dysfunctional DNA damage response (DDR). Cells with inefficient DDR are more vulnerable to therapeutic approaches that target DDR, thereby raising DNA damage to a threshold that triggers apoptosis. Here, we demonstrate that 2 Jurkat-derived cell lines with incorporated silent HIV-1 provirus show increases in DDR signaling that responds to formation of double strand DNA breaks (DSBs). We found that phosphorylation of histone H2AX on Ser139 (gamma-H2AX), a biomarker of DSBs, and phosphorylation of ATM at Ser1981, Chk2 at Thr68, and p53 at Ser15, part of signaling pathways associated with DSBs, are elevated in these cells. These results indicate a DDR defect even though the virus is latent. DDR-inducing agents, specifically high doses of nucleoside RT inhibitors (NRTIs), caused greater increases in gamma-H2AX levels in latently infected cells. Additionally, latently infected cells are more susceptible to long-term exposure to G-quadruplex stabilizing agents, and this effect is enhanced when the agent is combined with an inhibitor targeting DNA-PK, which is crucial for DSB repair and telomere maintenance. Moreover, exposing these cells to the cancer drug etoposide resulted in formation of DSBs at a higher rate than in un-infected cells. Similar effects of etoposide were also observed in population of primary memory T cells infected with latent HIV-1. Sensitivity to these agents highlights a unique vulnerability of latently infected cells, a new feature that could potentially be used in developing therapies to eliminate HIV-1 reservoirs.

  20. Edaravone Protects against Methylglyoxal-Induced Barrier Damage in Human Brain Endothelial Cells

    PubMed Central

    Tóth, Andrea E.; Walter, Fruzsina R.; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G.; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A.

    2014-01-01

    Background Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Methodology Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Principal Findings Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. Conclusion These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases. PMID:25033388

  1. Edaravone protects against methylglyoxal-induced barrier damage in human brain endothelial cells.

    PubMed

    Tóth, Andrea E; Walter, Fruzsina R; Bocsik, Alexandra; Sántha, Petra; Veszelka, Szilvia; Nagy, Lajos; Puskás, László G; Couraud, Pierre-Olivier; Takata, Fuyuko; Dohgu, Shinya; Kataoka, Yasufumi; Deli, Mária A

    2014-01-01

    Elevated level of reactive carbonyl species, such as methylglyoxal, triggers carbonyl stress and activates a series of inflammatory responses leading to accelerated vascular damage. Edaravone is the active substance of a Japanese medicine, which aids neurological recovery following acute brain ischemia and subsequent cerebral infarction. Our aim was to test whether edaravone can exert a protective effect on the barrier properties of human brain endothelial cells (hCMEC/D3 cell line) treated with methylglyoxal. Cell viability was monitored in real-time by impedance-based cell electronic sensing. The barrier function of the monolayer was characterized by measurement of resistance and flux of permeability markers, and visualized by immunohistochemistry for claudin-5 and β-catenin. Cell morphology was also examined by holographic phase imaging. Methylglyoxal exerted a time- and dose-dependent toxicity on cultured human brain endothelial cells: a concentration of 600 µM resulted in about 50% toxicity, significantly reduced the integrity and increased the permeability of the barrier. The cell morphology also changed dramatically: the area of cells decreased, their optical height significantly increased. Edaravone (3 mM) provided a complete protection against the toxic effect of methylglyoxal. Co-administration of edaravone restored cell viability, barrier integrity and functions of brain endothelial cells. Similar protection was obtained by the well-known antiglycating molecule, aminoguanidine, our reference compound. These results indicate for the first time that edaravone is protective in carbonyl stress induced barrier damage. Our data may contribute to the development of compounds to treat brain endothelial dysfunction in carbonyl stress related diseases.

  2. Genotoxic anti-cancer agents and their relationship to DNA damage, mitosis, and checkpoint adaptation in proliferating cancer cells.

    PubMed

    Swift, Lucy H; Golsteyn, Roy M

    2014-02-25

    When a human cell detects damaged DNA, it initiates the DNA damage response (DDR) that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies.

  3. Genotoxic Anti-Cancer Agents and Their Relationship to DNA Damage, Mitosis, and Checkpoint Adaptation in Proliferating Cancer Cells

    PubMed Central

    Swift, Lucy H.; Golsteyn, Roy M.

    2014-01-01

    When a human cell detects damaged DNA, it initiates the DNA damage response (DDR) that permits it to repair the damage and avoid transmitting it to daughter cells. Despite this response, changes to the genome occur and some cells, such as proliferating cancer cells, are prone to genome instability. The cellular processes that lead to genomic changes after a genotoxic event are not well understood. Our research focuses on the relationship between genotoxic cancer drugs and checkpoint adaptation, which is the process of mitosis with damaged DNA. We examine the types of DNA damage induced by widely used cancer drugs and describe their effects upon proliferating cancer cells. There is evidence that cell death caused by genotoxic cancer drugs in some cases includes exiting a DNA damage cell cycle arrest and entry into mitosis. Furthermore, some cells are able to survive this process at a time when the genome is most susceptible to change or rearrangement. Checkpoint adaptation is poorly characterised in human cells; we predict that increasing our understanding of this pathway may help to understand genomic instability in cancer cells and provide insight into methods to improve the efficacy of current cancer therapies. PMID:24573252

  4. Dose rate and repair effects on cell damage in earth orbit

    NASA Technical Reports Server (NTRS)

    Cucinotta, F. A.; Wilson, J. W.; Shinn, J. L.; Atwell, W.; Fong, M.

    1994-01-01

    Radiobiology experiments performed in space will encounter continuous exposures to the cosmic rays and fractionated exposures to trapped protons which accumulate to several hundred dose fractions in a few weeks. Using models of track structure and cellular kinetics combined with models of the radiation environment and radiation transport, we consider calculations of damage rates for cell cultures. Analysis of the role of repair mechanisms for space exposures for the endpoints of survival and transformation is emphasized.

  5. Hyperglycemia induced damage to mitochondrial respiration in renal mesangial and tubular cells: Implications for diabetic nephropathy.

    PubMed

    Czajka, Anna; Malik, Afshan N

    2016-12-01

    Damage to renal tubular and mesangial cells is central to the development of diabetic nephropathy (DN), a complication of diabetes which can lead to renal failure. Mitochondria are the site of cellular respiration and produce energy in the form of ATP via oxidative phosphorylation, and mitochondrial dysfunction has been implicated in DN. Since the kidney is an organ with high bioenergetic needs, we postulated that hyperglycemia causes damage to renal mitochondria resulting in bioenergetic deficit. The bioenergetic profiles and the effect of hyperglycemia on cellular respiration of human primary mesangial (HMCs) and proximal tubular cells (HK-2) were compared in normoglycemic and hyperglycemic conditions using the seahorse bio-analyzer. In normoglycemia, HK-2 had significantly lower basal, ATP-linked and maximal respiration rates, and lower reserve capacity compared to HMCs. Hyperglycemia caused a down-regulation of all respiratory parameters within 4 days in HK-2 but not in HMCs. After 8 days of hyperglycemia, down-regulation of respiratory parameters persisted in tubular cells with compensatory up-regulated glycolysis. HMCs had reduced maximal respiration and reserve capacity at 8 days, and by 12 days had compromised mitochondrial respiration despite which they did not enhance glycolysis. These data suggest that diabetes is likely to lead to a cellular deficit in ATP production in both cell types, although with different sensitivities, and this mechanism could significantly contribute to the cellular damage seen in the diabetic kidney. Prevention of diabetes induced damage to renal mitochondrial respiration may be a novel therapeutic approach for the prevention/treatment of DN. Copyright © 2016 The Authors. Published by Elsevier B.V. All rights reserved.

  6. Exogenous skeletal muscle satellite cells promote the repair of levator palpebrae superioris mechanical damage in rat.

    PubMed

    Ye, Lin; Yao, Yuanyuan; Guo, Hui; Peng, Yun

    2018-04-13

    Blepharoptosis is a drooping of the upper eyelid, usually due to dysfunction of the levator palpebrae superioris (LPS). Recently, skeletal muscle satellite cells (SSCs) have been reported to promote the repair of damaged skeletal muscle. This study aims to investigate the potential contribution of exogenous SSCs to the regeneration of mechanically damaged LPS. Thirty-two rats were randomly divided into four groups, including control group, SSCs-treated group, SSCs-treated injury group and non-treated injury group. After rats in injury groups were artificially lacerated on both the left and right LPS, HBBS (Hank's Balanced Salt Solution) containing SSCs was injected into upper eyelid tissue. After 7 days, the LPS muscle tissues were excised. In addition, skeletal muscle cells (SMCs) and SSCs were co-cultured for use as an in vitro model, and the protective effects of SSCs on cultured SMCs were also investigated. Histological staining revealed that exogenous SSCs repaired the damaged muscle fibers and attenuated the fibrosis of LPS, possibly due to the increased level of IGF-1. In contrast, the level of IL-1β, IL-6, TGF-β1 and Smad2/3(phospho-T8) were significantly reduced in the SSCs-treated group. The in vitro model using co-culture of skeletal muscle cells (SMCs) and SSCs also revealed an increased level of IGF-1 and reduced level of inflammatory factors, resulting in a better cell survival rate. This study found that exogenous SSCs can promote the repair of LPS mechanical damage and provides new insight into the development of novel therapeutic approaches for blepharoptosis.

  7. A simple model of proton damage in GaAs solar cells

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; Walker, G. H.; Outlaw, R. A.

    1982-01-01

    A simple proton damage model for GaAs solar cells is derived and compared to experimental values of change in short circuit currents. The recombination cross section associated with the defects was determined from the experimental comparison to be approximately 1.2 x 10 to the -13th power sq cm in fair agreement with values determined from the deep level transient spectroscopy technique.

  8. Contributions of DNA repair, cell cycle checkpoints and cell death to suppressing the DNA damage-induced tumorigenic behavior of Drosophila epithelial cells.

    PubMed

    Dekanty, A; Barrio, L; Milán, M

    2015-02-19

    When exposed to DNA-damaging agents, components of the DNA damage response (DDR) pathway trigger apoptosis, cell cycle arrest and DNA repair. Although failures in this pathway are associated with cancer development, the tumor suppressor roles of cell cycle arrest and apoptosis have recently been questioned in mouse models. Using Drosophila epithelial cells that are unable to activate the apoptotic program, we provide evidence that ionizing radiation (IR)-induced DNA damage elicits a tumorigenic behavior in terms of E-cadherin delocalization, cell delamination, basement membrane degradation and neoplasic overgrowth. The tumorigenic response of the tissue to IR is enhanced by depletion of Okra/DmRAD54 or spnA/DmRAD51--genes required for homologous recombination (HR) repair of DNA double-strand breaks in G2--and it is independent of the activity of Lig4, a ligase required for nonhomologous end-joining repair in G1. Remarkably, depletion of Grapes/DmChk1 or Mei-41/dATR-genes affecting DNA damage-induced cell cycle arrest in G2--compromised DNA repair and enhanced the tumorigenic response of the tissue to IR. On the contrary, DDR-independent lengthening of G2 had a positive impact on the dynamics of DNA repair and suppressed the tumorigenic response of the tissue to IR. Our results support a tumor suppressor role of apoptosis, DNA repair by HR and cell cycle arrest in G2 in simple epithelia subject to IR-induced DNA damage.

  9. The origin of the radiobiological damage in cells stored in cryostatic conditions

    NASA Astrophysics Data System (ADS)

    Dondi, D.; Buttafava, A.; Zeffiro, A.; Cherubini, R.; De Nadal, V.; Gerardi, S.; Faucitano, A.

    2012-09-01

    The radiation induced free radical damage in Chinese hamster lung fibroblast V-79 cells stored in DMEM culture medium containing 10% DMSO has been investigated by matrix EPR spectroscopy in connection with the H2O/DMSO binary phase diagram. A major part of the indirect effect is due to radicals from the DMSO·3H2O phase in the freezing medium, which are released on warming in the temperature range between 130 K and 160 K, that is, far below the eutectic melting temperature (210 K). The radicals trapped in the DMSO·3H2O phase react with oxygen above 160 K giving reactive oxygen species (ROS) of the type of peroxyl radicals. A lower limit yield of 10-15% was calculated for this conversion. Scavenging experiments with a stable nitroxyl radical (tempol) have demonstrated that part of the DMSO·3H2O radicals escape by mutual recombination on melting and are therefore available for inducing indirect cell damage. The same experiments performed with pure frozen water have shown that OH radicals are not available for inducing cell damage. The EPR measurements performed on H2O/DMSO frozen mixtures suggest that the radiation induced radical forming process does not change when passing to the low dose range below 1 Gy, in agreement with the linear model.

  10. Negative regulator of E2F transcription factors links cell cycle checkpoint and DNA damage repair.

    PubMed

    Wang, Lili; Chen, Hanchen; Wang, Chongyang; Hu, Zhenjie; Yan, Shunping

    2018-04-17

    DNA damage poses a serious threat to genome integrity and greatly affects growth and development. To maintain genome stability, all organisms have evolved elaborate DNA damage response mechanisms including activation of cell cycle checkpoints and DNA repair. Here, we show that the DNA repair protein SNI1, a subunit of the evolutionally conserved SMC5/6 complex, directly links these two processes in Arabidopsis SNI1 binds to the activation domains of E2F transcription factors, the key regulators of cell cycle progression, and represses their transcriptional activities. In turn, E2Fs activate the expression of SNI1 , suggesting that E2Fs and SNI1 form a negative feedback loop. Genetically, overexpression of SNI1 suppresses the phenotypes of E2F-overexpressing plants, and loss of E2F function fully suppresses the sni1 mutant, indicating that SNI1 is necessary and sufficient to inhibit E2Fs. Altogether, our study revealed that SNI1 is a negative regulator of E2Fs and plays dual roles in DNA damage responses by linking cell cycle checkpoint and DNA repair.

  11. Tumor induced hepatic myeloid derived suppressor cells can cause moderate liver damage.

    PubMed

    Eggert, Tobias; Medina-Echeverz, José; Kapanadze, Tamar; Kruhlak, Michael J; Korangy, Firouzeh; Greten, Tim F

    2014-01-01

    Subcutaneous tumors induce the accumulation of myeloid derived suppressor cells (MDSC) not only in blood and spleens, but also in livers of these animals. Unexpectedly, we observed a moderate increase in serum transaminases in mice with EL4 subcutaneous tumors, which prompted us to study the relationship of hepatic MDSC accumulation and liver injury. MDSC were the predominant immune cell population expanding in livers of all subcutaneous tumor models investigated (RIL175, B16, EL4, CT26 and BNL), while liver injury was only observed in EL4 and B16 tumor-bearing mice. Elimination of hepatic MDSC in EL4 tumor-bearing mice using low dose 5-fluorouracil (5-FU) treatment reversed transaminase elevation and adoptive transfer of hepatic MDSC from B16 tumor-bearing mice caused transaminase elevation indicating a direct MDSC mediated effect. Surprisingly, hepatic MDSC from B16 tumor-bearing mice partially lost their damage-inducing potency when transferred into mice bearing non damage-inducing RIL175 tumors. Furthermore, MDSC expansion and MDSC-mediated liver injury further increased with growing tumor burden and was associated with different cytokines including GM-CSF, VEGF, interleukin-6, CCL2 and KC, depending on the tumor model used. In contrast to previous findings, which have implicated MDSC only in protection from T cell-mediated hepatitis, we show that tumor-induced hepatic MDSC themselves can cause moderate liver damage.

  12. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage.

    PubMed

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-03-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (EN(KO)) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in EN(KO) mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. © 2015 The Authors. Published under the terms of the CC BY 4.0 license.

  13. Mechanical damage in a lithium-ion pouch cell under indentation loads

    NASA Astrophysics Data System (ADS)

    Luo, Hailing; Xia, Yong; Zhou, Qing

    2017-07-01

    The short circuit of lithium-ion batteries induced by mechanical abuse is a great concern in electric vehicle design. It remains a challenge to fully understand the nature of the mechanical damage process with the aim of improving battery crash safety. The present paper investigates the evolution of the damage process for a lithium-ion pouch cell under indentation by loading the cell to various force levels. A significant inflection point on the force-indentation curve is observed before the force peak. Post-mortem examinations indicate that the characteristic change in the local slope of the curve is related to the change occurring at the local interfaces, including three phenomena - formation of tight adhesion on the anode-separator interfaces, delamination in the separators and decoating of graphite particles from the anodes. Analysis of the fracture sequence at the onset of short circuit clearly shows that the number of short-circuited electrode pairs is equal to the number of anode layers adhered with delaminated separator material before fracture occurs. The experimental study in the present paper implies that the inflection point on the force-indentation curve may be an indicator of damage initiation inside pouch cells under indentation.

  14. Hepatic stellate cell-expressed endosialin balances fibrogenesis and hepatocyte proliferation during liver damage

    PubMed Central

    Mogler, Carolin; Wieland, Matthias; König, Courtney; Hu, Junhao; Runge, Anja; Korn, Claudia; Besemfelder, Eva; Breitkopf-Heinlein, Katja; Komljenovic, Dorde; Dooley, Steven; Schirmacher, Peter; Longerich, Thomas; Augustin, Hellmut G

    2015-01-01

    Liver fibrosis is a reversible wound-healing response to injury reflecting the critical balance between liver repair and scar formation. Chronic damage leads to progressive substitution of liver parenchyma by scar tissue and ultimately results in liver cirrhosis. Stromal cells (hepatic stellate cells [HSC] and endothelial cells) have been proposed to control the balance between liver fibrosis and regeneration. Here, we show that endosialin, a C-type lectin, expressed in the liver exclusively by HSC and portal fibroblasts, is upregulated in liver fibrosis in mouse and man. Chronic chemically induced liver damage resulted in reduced fibrosis and enhanced hepatocyte proliferation in endosialin-deficient (ENKO) mice. Correspondingly, acute-liver-damage-induced hepatocyte proliferation (partial hepatectomy) was increased in ENKO mice. A candidate-based screen of known regulators of hepatocyte proliferation identified insulin-like growth factor 2 (IGF2) as selectively endosialin-dependent hepatocyte mitogen. Collectively, the study establishes a critical role of HSC in the reciprocal regulation of fibrogenesis vs. hepatocyte proliferation and identifies endosialin as a therapeutic target in non-neoplastic settings. PMID:25680861

  15. Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse

    PubMed Central

    2017-01-01

    We report functional and structural evidence for GluA2-lacking Ca2+-permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss. SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca2+-permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca2+ accumulation and swelling that can be prevented by blocking CP-AMPARs. We

  16. TGF-β1 accelerates the DNA damage response in epithelial cells via Smad signaling

    SciTech Connect

    Lee, Jeeyong; Kim, Mi-Ra; Kim, Hyun-Ji

    2016-08-05

    The evidence suggests that transforming growth factor-beta (TGF-β) regulates the DNA-damage response (DDR) upon irradiation, and we previously reported that TGF-β1 induced DNA ligase IV (Lig4) expression and enhanced the nonhomologous end-joining repair pathway in irradiated cells. In the present study, we investigated the effects of TGF-β1 on the irradiation-induced DDRs of A431 and HaCaT cells. Cells were pretreated with or without TGF-β1 and irradiated. At 30 min post-irradiation, DDRs were detected by immunoblotting of phospho-ATM, phospho-Chk2, and the presence of histone foci (γH2AX). The levels of all three factors were similar right after irradiation regardless of TGF-β1 pretreatment. However, theymore » soon thereafter exhibited downregulation in TGF-β1-pretreated cells, indicating the acceleration of the DDR. Treatment with a TGF-β type I receptor inhibitor (SB431542) or transfections with siRNAs against Smad2/3 or DNA ligase IV (Lig4) reversed this acceleration of the DDR. Furthermore, the frequency of irradiation-induced apoptosis was decreased by TGF-β1 pretreatment in vivo, but this effect was abrogated by SB431542. These results collectively suggest that TGF-β1 could enhance cell survival by accelerating the DDR via Smad signaling and Lig4 expression. -- Highlights: •TGF-β1 pretreatment accelerates γ-radiation-induced DNA damage response. •TGF-β1-accelerated DNA damage response is dependent on Smad signaling and DNA Ligase IV. •TGF-β1 pretreatment protects epithelial cells from γ-radiation in vivo.« less

  17. Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells

    PubMed Central

    Rai, Prashant; Parrish, Marcus; Tay, Ian Jun Jie; Li, Na; Ackerman, Shelley; He, Fang; Kwang, Jimmy; Chow, Vincent T.; Engelward, Bevin P.

    2015-01-01

    Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia. PMID:26080406

  18. Streptococcus pneumoniae secretes hydrogen peroxide leading to DNA damage and apoptosis in lung cells.

    PubMed

    Rai, Prashant; Parrish, Marcus; Tay, Ian Jun Jie; Li, Na; Ackerman, Shelley; He, Fang; Kwang, Jimmy; Chow, Vincent T; Engelward, Bevin P

    2015-06-30

    Streptococcus pneumoniae is a leading cause of pneumonia and one of the most common causes of death globally. The impact of S. pneumoniae on host molecular processes that lead to detrimental pulmonary consequences is not fully understood. Here, we show that S. pneumoniae induces toxic DNA double-strand breaks (DSBs) in human alveolar epithelial cells, as indicated by ataxia telangiectasia mutated kinase (ATM)-dependent phosphorylation of histone H2AX and colocalization with p53-binding protein (53BP1). Furthermore, results show that DNA damage occurs in a bacterial contact-independent fashion and that Streptococcus pyruvate oxidase (SpxB), which enables synthesis of H2O2, plays a critical role in inducing DSBs. The extent of DNA damage correlates with the extent of apoptosis, and DNA damage precedes apoptosis, which is consistent with the time required for execution of apoptosis. Furthermore, addition of catalase, which neutralizes H2O2, greatly suppresses S. pneumoniae-induced DNA damage and apoptosis. Importantly, S. pneumoniae induces DSBs in the lungs of animals with acute pneumonia, and H2O2 production by S. pneumoniae in vivo contributes to its genotoxicity and virulence. One of the major DSBs repair pathways is nonhomologous end joining for which Ku70/80 is essential for repair. We find that deficiency of Ku80 causes an increase in the levels of DSBs and apoptosis, underscoring the importance of DNA repair in preventing S. pneumoniae-induced genotoxicity. Taken together, this study shows that S. pneumoniae-induced damage to the host cell genome exacerbates its toxicity and pathogenesis, making DNA repair a potentially important susceptibility factor in people who suffer from pneumonia.

  19. Functionally and morphologically damaged mitochondria observed in auditory cells under senescence-inducing stress.

    PubMed

    Kamogashira, Teru; Hayashi, Ken; Fujimoto, Chisato; Iwasaki, Shinichi; Yamasoba, Tatsuya

    2017-01-01

    We aimed at determining the mitochondrial function in premature senescence model of auditory cells. Short exposure to H 2 O 2 (1 h, 0.1 mM) induced premature cellular senescence in House Ear Institute-Organ of Corti 1 auditory cells. The transmission electron microscopy analysis revealed that damaged mitochondria and autophagosomes containing dense organelles appeared in the auditory cells after short exposure to H 2 O 2 . The branch and junction parameters of the skeletonized image of the mitochondria were found to decrease significantly in H 2 O 2 -treated cells. A branched reticulum of tubules was poorly formed, featuring coexistence of numerous tiny clusters along with few relatively large entities in the H 2 O 2 -treated cells. In terms of bioenergetics, H 2 O 2 -treatment led to the dose-dependent decrease in mitochondrial membrane potential in the auditory cells. The fragmented mitochondria (fusion < fission) were in a low potential. In addition, the potential of hyperfused mitochondria (fusion > fission) was slightly lower than the control cells. The short-time exposure of live auditory cells to H 2 O 2 damaged the mitochondrial respiratory capacity without any effect on the baseline ATP production rates. The vulnerability of the mitochondrial membrane potential to the uncoupling reagent was increased after H 2 O 2 treatment. Our findings indicated that the mitochondrial dysfunction due to the decline in the O 2 consumption rate should be the first event of premature senescence process in the auditory cells, resulting in the imbalance of mitochondrial fusion/fission and the collapse of the mitochondrial network.

  20. Renal-protective and ameliorating impacts of omega-3 fatty acids against aspartame damaged MDCK cells.

    PubMed

    Pandurangan, Muthuraman; Enkhtaivan, Gansukh; Veerappan, Muthuviveganandavel; Mistry, Bhupendra; Patel, Rahul; Moon, So Hyun; Nagajyothi, Patnamsetty Chidanandha; Kim, Doo Hwan

    2017-11-01

    Aspartame is widely used artificial sweeteners as food additives. Several researchers have pointed that the controversial report on the use of aspartame over more than decades. Omega-3 fatty acids are essential and unsaturated fatty acids, and it plays a remarkable role in vision, intelligence, neural development, and metabolism of neurotransmitters. Therefore, the present study was aimed to investigate the effect of omega-3 fatty acids on aspartame treated renal cells. Experimental groups were divided into three such as sham control, aspartame treated, and aspartame with omega-3 fatty acids. Cell viability was determined by sulforhodamine-b assay and flow cytometric analysis. The experimental results showed that the aspartame induced altered cell viability were reduced following treatment of aspartame with omega-3 fatty acids. Altered cell morphology was recovered by omega-3 fatty acids. DNA damage appeared in the highest concentration of aspartame used in this study. DNA damage characteristics such as comet tail and tiny head sections did not appear in the omega-3 fatty acids treated cells. Several microvilli and vesicular structures were found in aspartame treated cells. Altered morphology such as rounding, microvilli, and formation of dome-like structures did not appear in the omega-3 fatty acids with aspartame treated cells. Caspase-3 mRNA and protein expression were increased in aspartame treated cells, and these levels were reduced following omega-3 fatty acids treatment. Taking all these data together, it is suggested that the omega-3 fatty acids may be a therapeutic agent to reduce the aspartame induced biochemical and morphological alterations in normal renal cells. © 2017 BioFactors, 43(6):847-857, 2017. © 2017 International Union of Biochemistry and Molecular Biology.

  1. Licochalcone A-Induced Human Bladder Cancer T24 Cells Apoptosis Triggered by Mitochondria Dysfunction and Endoplasmic Reticulum Stress

    PubMed Central

    Yuan, Xuan; Li, Defang; Zhao, Hong; Jiang, Jiangtao; Wang, Penglong; Ma, Xiaoyi; Sun, Xiling; Zheng, Qiusheng

    2013-01-01

    Licochalcone A (LCA), a licorice chalconoid, is considered to be a bioactive agent with chemopreventive potential. This study investigated the mechanisms involved in LCA-induced apoptosis in human bladder cancer T24 cells. LCA significantly inhibited cells proliferation, increased reactive oxygen species (ROS) levels, and caused T24 cells apoptosis. Moreover, LCA induced mitochondrial dysfunction, caspase-3 activation, and poly-ADP-ribose polymerase (PARP) cleavage, which displayed features of mitochondria-dependent apoptotic signals. Besides, exposure of T24 cells to LCA triggered endoplasmic reticulum (ER) stress; as indicated by the enhancement in 78 kDa glucose-regulated protein (GRP 78), growth arrest and DNA damage-inducible gene 153/C/EBP homology protein (GADD153/CHOP) expression, ER stress-dependent apoptosis is caused by the activation of ER-specific caspase-12. All the findings from our study suggest that LCA initiates mitochondrial ROS generation and induces oxidative stress that consequently causes T24 cell apoptosis via the mitochondria-dependent and the ER stress-triggered signaling pathways. PMID:23936805

  2. Evaluation of various glyphosate concentrations on DNA damage in human Raji cells and its impact on cytotoxicity.

    PubMed

    Townsend, Michelle; Peck, Connor; Meng, Wei; Heaton, Matthew; Robison, Richard; O'Neill, Kim

    2017-04-01

    Glyphosate is a highly used active compound in agriculturally based pesticides. The literature regarding the toxicity of glyphosate to human cells has been highly inconsistent. We studied the resulting DNA damage and cytotoxicity of various glyphosate concentrations on human cells to evaluate DNA damaging potential. Utilizing human Raji cells, DNA damage was quantified using the comet assay, while cytotoxicity was further analyzed using MTT viability assays. Several glyphosate concentrations were assessed, ranging from 15 mM to 0.1 μM. We found that glyphosate treatment is lethal to Raji cells at concentrations above 10 mM, yet has no cytotoxic effects at concentrations at or below 100 μM. Treatment concentrations of 1 mM and 5 mM induce statistically significant DNA damage to Raji cells following 30-60 min of treatment, however, cells show a slow recovery from initial damage and cell viability is unaffected after 2 h. At these same concentrations, cells treated with additional compound did not recover and maintained high levels of DNA damage. While the cytotoxicity of glyphosate appears to be minimal for physiologically relevant concentrations, the compound has a definitive cytotoxic nature in human cells at high concentrations. Our data also suggests a mammalian metabolic pathway for the degradation of glyphosate may be present. Copyright © 2017 Elsevier Inc. All rights reserved.

  3. Chromosomal Damage and Apoptosis in Exfoliated Buccal Cells from Individuals with Oral Cancer

    PubMed Central

    Dórea, Lavínia Tércia Magalhães; Meireles, José Roberto Cardoso; Lessa, Júlia Paula Ramos; Oliveira, Márcio Campos; de Bragança Pereira, Carlos Alberto; Polpo de Campos, Adriano; Cerqueira, Eneida de Moraes Macílio

    2012-01-01

    This study aimed to investigate cytological abnormalities indicative of chromosome damage (micronuclei) and apoptosis (karyorrhexis, pyknosis, and condensed chromatin) in exfoliated cells from the buccal mucosa of patients with oral cancer and control subjects. The sample included twenty individuals with oral cancer and forty individuals with normal buccal mucosa. Material was collected from the cheek epithelium in areas with lesions and areas without abnormalities. A minimum of one thousand cells was analyzed. Micronuclei were found significantly more frequently in cells collected from lesions than in cells from normal areas, independent of the presence/absence of cancer (P < 0.0001). They were also significantly more frequent in smokers and in mouthwash users (P < 0.0001). Apoptosis occurred significantly less frequently in individuals with oral cancer (P < 0.0001). These results show that oral cancer is associated with higher frequency of chromosomal damage and suggest that apoptosis is compromised in the buccal cells of individuals with this kind of neoplasia. PMID:22315605

  4. Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells.

    PubMed

    Cellai, Filippo; Munnia, Armelle; Viti, Jessica; Doumett, Saer; Ravagli, Costanza; Ceni, Elisabetta; Mello, Tommaso; Polvani, Simone; Giese, Roger W; Baldi, Giovanni; Galli, Andrea; Peluso, Marco E M

    2017-04-29

    Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3 H )-one deoxyguanosine (M₁dG) and 8-oxo-7,8-dihydro-2'-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe₃O₄-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32 P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 μg/mL of Fe₃O₄-NPs. Significant dose-response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death.

  5. Magnetic Hyperthermia and Oxidative Damage to DNA of Human Hepatocarcinoma Cells

    PubMed Central

    Cellai, Filippo; Munnia, Armelle; Viti, Jessica; Doumett, Saer; Ravagli, Costanza; Ceni, Elisabetta; Mello, Tommaso; Polvani, Simone; Giese, Roger W.; Baldi, Giovanni; Galli, Andrea; Peluso, Marco E. M.

    2017-01-01

    Nanotechnology is addressing major urgent needs for cancer treatment. We conducted a study to compare the frequency of 3-(2-deoxy-β-d-erythro-pentafuranosyl)pyrimido[1,2-α]purin-10(3H)-one deoxyguanosine (M1dG) and 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxodG) adducts, biomarkers of oxidative stress and/or lipid peroxidation, on human hepatocarcinoma HepG2 cells exposed to increasing levels of Fe3O4-nanoparticles (NPs) versus untreated cells at different lengths of incubations, and in the presence of increasing exposures to an alternating magnetic field (AMF) of 186 kHz using 32P-postlabeling. The levels of oxidative damage tended to increase significantly after ≥24 h of incubations compared to controls. The oxidative DNA damage tended to reach a steady-state after treatment with 60 μg/mL of Fe3O4-NPs. Significant dose–response relationships were observed. A greater adduct production was observed after magnetic hyperthermia, with the highest amounts of oxidative lesions after 40 min exposure to AMF. The effects of magnetic hyperthermia were significantly increased with exposure and incubation times. Most important, the levels of oxidative lesions in AMF exposed NP treated cells were up to 20-fold greater relative to those observed in nonexposed NP treated cells. Generation of oxidative lesions may be a mechanism by which magnetic hyperthermia induces cancer cell death. PMID:28468256

  6. Tiron, a ROS scavenger, protects human lung cancer Calu-6 cells against antimycin A-induced cell death.

    PubMed

    Han, Yong Hwan; Park, Woo Hyun

    2009-01-01

    Antimycin A (AMA) inhibits the mitochondrial electron transport between cytochromes b and c. However, the relationship between AMA and lung cancer cells is poorly understood. In this study, we investigated the involvement of reactive oxygen species (ROS) and glutathione (GSH) in AMA-treated lung cancer Calu-6 cell death. Treatment with AMA reduced cell viability in a dose-dependent manner for 72 h. The intracellular ROS levels were decreased in Calu-6 cells treated with low doses of AMA (10, 25 or 50 microM) at 72 h. However, the levels increased in cells treated with a high dose of 100 microM AMA. Levels of O2.- were significantly increased in AMA-treated cells at 72 h. The increases in ROS levels including O2.- in AMA-treated cells were observed within 10 min. Treatment with AMA reduced the intracellular GSH content. SOD activity was up-regulated in AMA-treated Calu-6 cells at 72 h. However, catalase activity was down-regulated by AMA. Treatment with tiron, a ROS scavenger, reduced the intracellular ROS levels, which were associated with a partial reduction of apoptosis. Treatment with exogenous SOD and catalase significantly inhibited loss of the mitochondrial transmembrane potential (DeltaPsim) in AMA-treated Calu-6 cells. In conclusion, our results suggest that the changes of intracellular ROS and GSH affect apoptosis in AMA-treated Calu-6 cells.

  7. Cyclosporin A-induced hydrogen peroxide synthesis by cultured human mesangial cells is blocked by exogenous antioxidants.

    PubMed

    Pérez de Lema, G; Arribas, I; Prieto, A; Parra, T; de Arriba, G; Rodríguez-Puyol, D; Rodríguez-Puyol, M

    1998-01-01

    Cyclosporin A (CsA) is the immunosupressor most widely used in transplanted patients for preventing organ rejection, but it has some toxic side effects in vascular beds and kidney. The purpose of this work was to study if H2O2, a reactive oxygen species, is involved in the CsA-induced toxic effects on kidney in vitro. Human mesangial cells (HMC) in culture were incubated in presence of CsA (10[-5]-10[-8]M) and H2O2 was measured by flow cytometry. The specificity of the probe used in this method was demonstrated as fluorescence was not detected when superoxide anion generated through a Xanthine-Xanthine oxidase system was present, but fluorescence was noted when H2O2 was present in the incubation medium, both directly and after addition of superoxide dismutase to the medium thus promoting H2O2 synthesis. CsA induced a significant dose and time-response increased H2O2 synthesis by cultured HMC. This increase appeared 5 min after CsA addition, being maximal between 15-45 min at CsA concentration of 10(-7)M. When HMC were preincubated with antioxidants as vitamin E or selenium, the CsA-induced H2O2 production was partially blocked. In addition, selenium also induced an increased activity of glutathion peroxidase in HMC after 24 hours of incubation, suggesting that it exerted its H2O2 scavenging action through the modulation of the activity of this enzyme.

  8. Damage and Recovery of Hair Cells in Fish Canal (But Not Superficial) Neuromasts after Gentamicin Exposure

    NASA Technical Reports Server (NTRS)

    Song, Jiakun; Yan, Hong Young; Popper, Arthur N.

    1995-01-01

    Recent evidence demonstrating the presence of two types of sensory hair cells in the ear of a telcost fish (Astronotus ocellatus, the oscar) indicates that hair cell heterogeneity may exist not only in amniotic vertebrates but also in anamniotes. Here we report that a similar heterogeneity between hair cell types may also occur in the other mechanosensory organ of the oscar, the lateral line. We exposed oscars to the aminoglycoside (ototoxic) antibiotic gentamicin sulfate and found damaged sensory hair cells in one class of the lateral line receptors, the canal neuromasts, but not in the other class, the superficial neuromasts. This effect was not due to the canal environment. Moreover, new ciliary bundles on hair cells of the canal neuromasts were found after, and during, gentamicin exposure. The pattern of hair cell destruction and recovery in canal neuromasts is similar to that of type 1-like hair cells found in the striolar region of the utricle and lagena of the oscar after gentamicin treatment. These results suggest that the hair cells in the canal and superficial neuromasts may be similar to type 1-like and type 2 hair cells, respectively, in the fish ear.

  9. Vitrification of bovine blastocysts produced in vitro inflicts selective damage to the inner cell mass.

    PubMed

    Gómez, E; Muñoz, M; Rodríguez, A; Caamaño, J N; Facal, N; Díez, C

    2009-04-01

    In contrast to the embryos derived from live animals, the embryos produced in vitro undergo increased damage and reduced survival after cryopreservation, particularly when produced with serum. In medium containing serum, retinoic acid increases cell numbers in the inner cell mass and the trophectoderm without altering their relative proportions in the bovine blastocyst. In this work, in medium without serum, we analyzed the contribution of retinoic acid to the development of blastocyst and survival to vitrification, and found a strong cell reduction in the inner mass when compared to the trophectoderm. Day-6 in vitro-produced morulae were treated for 24 h with retinoic acid (0.7 and 1.4 microm) and subsequently cultured without additives for a further 24 h period. Day-8 blastocyst production and cell counts in hatched blastocysts were unaffected by retinoic acid. However, Day-7 expanded, vitrified embryos produced with retinoic acid 1.4 microm survived at lower rates than controls when cultured after warming. Vitrification greatly reduced cell numbers in the inner mass (p < 0.0001), while cells in the trophectoderm remained unaltered. Differential cell counts analysis in blastocysts should be taken up to replace unspecific determination of total cells to appreciate substantial modifications in their exact terms. The strong reduction we found in the inner cell mass could explain why in vitro survival to cryopreservation is sometimes scarcely informative on the viability of the embryo after transfer to recipients.

  10. Relationships between DNA damage and the survival of murine bone marrow cells irradiated in situ.

    PubMed

    Petrovecki, M; Prager, A; Terry, N H; Murray, D

    1994-06-01

    The relationships between DNA damage and the survival of murine bone marrow cells irradiated in situ were examined. Cell survival was assayed by the ability of bone marrow cells from irradiated mice to form colonies in vitro (CFU-C). DNA double-strand breaks (DSBs) were measured by neutral (nondenaturing) filter elution and pulsed-field gel electrophoresis (PFGE). Double-strand breaks were measured in the proliferating bone marrow cells, identified by injecting the mice with [3H]dThd at various times before gamma irradiation, as a model of the behavior of the radiosensitive target cells. To assess how the DNA lesions measured using these techniques correlated with cell killing, the effect of the radioprotective agent WR-2721 on the induction of DSBs in proliferating bone marrow cells was compared with its effect on CFU-C survival. WR-2721 protected against the killing of both granulocyte-macrophage and erythroid burst-forming CFU-C by a factor of about 2. In contrast, little (1.2-fold) protection was observed in the PFGE assay at radiation doses between 5 and 20 Gy. Similarly, at the lowest dose studied (5 Gy) there was little protection against DSBs as measured by neutral elution; only after doses of between 10 and 30 Gy was significant protection observed. Thus the previously reported predictive relationship between DSBs and cell survival in vitro does not appear to extend directly to murine bone marrow cells irradiated in vivo.

  11. DNA damage by carbon nanotubes using the single cell gel electrophoresis technique.

    PubMed

    Zeni, Olga; Scarfì, Maria Rosaria

    2010-01-01

    The single-cell gel electrophoresis (SCGE) or comet assay is a simple and sensitive method for quantitatively measuring DNA breakage and repair in individual cells. It can be applied to proliferating and non-proliferating cells and cells of those tissues, which are the first contact sites for mutagenic/carcinogenic substances. In this technique, cells are embedded in agarose, lysed, subjected to electrophoresis, and stained with a fluorescent DNA-binding dye. Cells with increased DNA damage display increased DNA migration from the nucleus toward the anode, which resembles the shape of a comet. The migration is observed by fluorescence microscopy after staining with a fluorescent DNA-binding dye, and the intensity of the comet tail reflects the number of DNA breaks. The assay is performed in almost all eukaryotic cells and has applications in many fields, including genetic toxicology, biomonitoring, ecotoxicology, medical, and nutritional research. The assay is a very sensitive tool to investigate the effect of carbon nanotubes on DNA of human cells in vitro. This chapter describes a procedure to perform the comet assay, in its alkaline version, on cell cultures treated with carbon nanotubes.

  12. Quantification of DNA damage products by gas chromatography tandem mass spectrometry in lung cell lines and prevention effect of thyme antioxidants on oxidative induced DNA damage.

    PubMed

    Aybastıer, Önder; Dawbaa, Sam; Demir, Cevdet; Akgün, Oğuzhan; Ulukaya, Engin; Arı, Ferda

    2018-03-01

    Lung cancer has a high treatment cost and poor prognosis in comparison to other types of cancers. This work was involved in studying oxidative DNA base damage inhibition. Accordingly, standard carvacrol, thymol, thymoquinone with water and water-methanol extract of thyme (Origanum vulgare L. subsp. hirtum (link.) Ietswaart), thyme oil and thyme water were prepared and investigated for their efficacy to inhibit DNA oxidative damage formed by H 2 O 2 in malignant lung cells (A549). The antioxidant capacity by ABTS assay was 271.73 ± 11.45 mg trolox equivalent/mL for thyme oil. HPLC analysis was carried out to determine the contents of different thyme extracts, results showing the presence of carvacrol, thymol, protocatechuic acid, caffeic acid, epicatechin and rosmarinic acid in water and water-methanol extracts while only carvacrol and thymol were found in thyme oil and thyme water. After DNA isolation from the cultured cells, the formed oxidative induced DNA damage products were analysed using GC-MS/MS. It was proven that the antioxidants in the cell culture media have succeeded to inhibit oxidative DNA base damage. Thymoquinone was shown to be the best protectant antioxidant among other antioxidants against the formation of oxidative DNA damage, whereas water-methanol extract of thyme was the best among the plant-sourced samples. Thymoquinone and thyme water-methanol extract were investigated for their efficacy on cultured healthy lung cells (BEAS-2B), and it was proven that they are efficient in protection against the oxidation of DNA of healthy lung cells too. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Frost induced damages within porous materials - from concrete technology to fuel cells technique

    NASA Astrophysics Data System (ADS)

    Palecki, Susanne; Gorelkov, Stanislav; Wartmann, Jens; Heinzel, Angelika

    2017-12-01

    Porous media like concrete or layers of membrane electrode assemblies (MEA) within fuel cells are affected by a cyclic frost exposure due to different damage mechanisms which could lead to essential degradation of the material. In general, frost damages can only occur in case of a specific material moisture content. In fuel cells, residual water is generally available after shut down inside the membrane i.e. the gas diffusion layer (GDL). During subsequent freezing, this could cause various damage phenomena such as frost heaves and delamination effects of the membrane electrode assembly, which depends on the location of pore water and on the pore structure itself. Porous materials possess a pore structure that could range over several orders of magnitudes with different properties and freezing behaviour of the pore water. Latter can be divided into macroscopic, structured and pre-structured water, influenced by surface interactions. Therefore below 0 °C different water modifications can coexist in a wide temperature range, so that during frost exposure a high amount of unfrozen and moveable water inside the pore system is still available. This induces transport mechanisms and shrinkage effects. The physical basics are similar for porous media. While the freezing behaviour of concrete has been studied over decades of years, in order to enhance the durability, the know-how about the influence of a frost attack on fuel cell systems is not fully understood to date. On the basis of frost damage models for concrete structures, an approach to describe the impact of cyclic freezing and thawing on membrane electrode assemblies has been developed within this research work. Major aim is beyond a better understanding of the frost induced mechanisms, the standardization of a suitable test procedure for the assessment of different MEA materials under such kind of attack. Within this contribution first results will be introduced.

  14. JP-8 jet fuel-induced DNA damage in H4IIE rat hepatoma cells.

    PubMed

    Grant, G M; Jackman, S M; Kolanko, C J; Stenger, D A

    2001-01-25

    We investigated the genotoxicity of middle distillate jet fuel, Jet Propulsion 8 (JP-8), on H4IIE rat hepatoma cells in vitro. DNA damage was evaluated using the comet (single cell gel electrophoresis) assay. Cells were exposed for 4h to JP-8 (solubilized in ethanol (EtOH) at 0.1% (v/v)) to concentrations ranging from 1 to 20microg/ml. Exposure to JP-8 resulted in an overall increase in mean comet tail moments ranging from 0.74+/-0.065 (0.1% EtOH control) to 3.13+/-0.018,4.36+/-0.32,5.40+/-0.29,7.70+/-0.52 and 11.23+/-0.77 for JP-8 concentrations 3, 5, 10, 15 and 20microg/ml, respectively. Addition of DNA repair inhibitors hydroxyurea (HU) and cytosine arabinoside (Ara-C) to cell culture with JP-8 resulted in accumulation of DNA damage strand breaks and increase in comet tail length. Inclusion of 4mM HU and 40microM Ara-C with 3, 5, 10 and 20microg/ml JP-8 concentrations resulted in increased mean tail moments to 5.94+/-0.43,10.12+/-0.72,17.03+/-0.96,and29.25+/-1.55. JP-8, in the concentrations used in this study, did not result in cytotoxicity or significant apoptosis, as measured using the terminal deoxynucleotidyl transferase (TDT)-mediated dUTP-X nick end labeling (TUNEL) assay. These results demonstrate that relevant exposures to JP-8 result in DNA damage to H4IIE cells, and suggest that DNA repair is involved in mitigating these effects.

  15. Mycobacterium tuberculosis Ku can bind to nuclear DNA damage and sensitize mammalian cells to bleomycin sulfate.

    PubMed

    Castore, Reneau; Hughes, Cameron; Debeaux, Austin; Sun, Jingxin; Zeng, Cailing; Wang, Shih-Ya; Tatchell, Kelly; Shi, Runhua; Lee, Kyung-Jong; Chen, David J; Harrison, Lynn

    2011-11-01

    Radiotherapy and chemotherapy are effective cancer treatments due to their ability to generate DNA damage. The major lethal lesion is the DNA double-strand break (DSB). Human cells predominantly repair DSBs by non-homologous end joining (NHEJ), which requires Ku70, Ku80, DNA-PKcs, DNA ligase IV and accessory proteins. Repair is initiated by the binding of the Ku heterodimer at the ends of the DSB and this recruits DNA-PKcs, which initiates damage signaling and functions in repair. NHEJ also exists in certain types of bacteria that have dormant phases in their life cycle. The Mycobacterium tuberculosis Ku (Mt-Ku) resembles the DNA-binding domain of human Ku but does not have the N- and C-terminal domains of Ku70/80 that have been implicated in binding mammalian NHEJ repair proteins. The aim of this work was to determine whether Mt-Ku could be used as a tool to bind DSBs in mammalian cells and sensitize cells to DNA damage. We generated a fusion protein (KuEnls) of Mt-Ku, EGFP and a nuclear localization signal that is able to perform bacterial NHEJ and hence bind DSBs. Using transient transfection, we demonstrated that KuEnls is able to bind laser damage in the nucleus of Ku80-deficient cells within 10 sec and remains bound for up to 2 h. The Mt-Ku fusion protein was over-expressed in U2OS cells and this increased the sensitivity of the cells to bleomycin sulfate. Hydrogen peroxide and UV radiation do not predominantly produce DSBs and there was little or no change in sensitivity to these agents. Since in vitro studies were unable to detect binding of Mt-Ku to DNA-PKcs or human Ku70/80, this work suggests that KuEnls sensitizes cells by binding DSBs, preventing human NHEJ. This study indicates that blocking or decreasing the binding of human Ku to DSBs could be a method for enhancing existing cancer treatments.

  16. Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes.

    PubMed

    Tran, Thai Q; Ishak Gabra, Mari B; Lowman, Xazmin H; Yang, Ying; Reid, Michael A; Pan, Min; O'Connor, Timothy R; Kong, Mei

    2017-11-01

    Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer.

  17. Glutamine deficiency induces DNA alkylation damage and sensitizes cancer cells to alkylating agents through inhibition of ALKBH enzymes

    PubMed Central

    Tran, Thai Q.; Ishak Gabra, Mari B.; Lowman, Xazmin H.; Yang, Ying; Reid, Michael A.; Pan, Min; O’Connor, Timothy R.

    2017-01-01

    Driven by oncogenic signaling, glutamine addiction exhibited by cancer cells often leads to severe glutamine depletion in solid tumors. Despite this nutritional environment that tumor cells often experience, the effect of glutamine deficiency on cellular responses to DNA damage and chemotherapeutic treatment remains unclear. Here, we show that glutamine deficiency, through the reduction of alpha-ketoglutarate, inhibits the AlkB homolog (ALKBH) enzymes activity and induces DNA alkylation damage. As a result, glutamine deprivation or glutaminase inhibitor treatment triggers DNA damage accumulation independent of cell death. In addition, low glutamine-induced DNA damage is abolished in ALKBH deficient cells. Importantly, we show that glutaminase inhibitors, 6-Diazo-5-oxo-L-norleucine (DON) or CB-839, hypersensitize cancer cells to alkylating agents both in vitro and in vivo. Together, the crosstalk between glutamine metabolism and the DNA repair pathway identified in this study highlights a potential role of metabolic stress in genomic instability and therapeutic response in cancer. PMID:29107960

  18. The photodynamic action of pheophorbide a induces cell death through oxidative stress in Leishmania amazonensis.

    PubMed

    Miranda, Nathielle; Volpato, Hélito; da Silva Rodrigues, Jean Henrique; Caetano, Wilker; Ueda-Nakamura, Tânia; de Oliveira Silva, Sueli; Nakamura, Celso Vataru

    2017-09-01

    Leishmaniasis is a disease caused by hemoflagellate protozoa, affecting millions of people worldwide. The difficulties of treating patients with this parasitosis include the limited efficacy and many side effects of the currently available drugs. Therefore, the search for new compounds with leishmanicidal action is necessary. Photodynamic therapy has been studied in the medical field because of its selectivity, utilizing a combination of visible light, a photosensitizer compound, and singlet oxygen to reach the area of treatment. The continued search for selective alternative treatments and effective targets that impact the parasite and not the host are fundamentally important for the development of new drugs. Pheophorbide a is a photosensitizer that may be promising for the treatment of leishmaniasis. The present study evaluated the in vitro biological effects of pheophorbide a and its possible mechanisms of action in causing cell death in L. amazonensis. Pheophorbide a was active against promastigote and amastigote forms of the parasite. After treatment, we observed ultrastructural alterations in this protozoan. We also observed changes in promastigote macromolecules and organelles, such as loss of mitochondrial membrane potential [∆Ψ m ], lipid peroxidation, an increase in lipid droplets, DNA fragmentation, phosphatidylserine exposure, an increase in caspase-like activity, oxidative imbalance, and a decrease in antioxidant defense systems. These findings suggest that cell death occurred through apoptosis. The mechanism of cell death in intracellular amastigotes appeared to involve autophagy, in which we clearly observed an increase in reactive oxygen species, a compromised ∆Ψ m , and an increase in the number of autophagic vacuoles. The present study contributes to the development of new photosensitizers against L. amazonensis. We also elucidated the mechanism of action of pheophorbide a, mainly in intracellular amastigotes, which is the most clinically

  19. Circulating nucleic acids damage DNA of healthy cells by integrating into their genomes

    PubMed Central

    Mittra, Indraneel; Khare, Naveen Kumar; Raghuram, Gorantla Venkata; Chaubal, Rohan; Khambatti, Fatema; Gupta, Deepika; Gaikwad, Ashwini; Prasannan, Preeti; Singh, Akshita; Iyer, Aishwarya; Singh, Ankita; Upadhyay, Pawan; Nair, Naveen Kumar; Mishra, Pradyumna Kumar; Dutt, Amit

    2018-01-01

    Whether nucleic acids that circulate in blood have any patho-physiological functions in the host have not been explored. We report here that far from being inert molecules, circulating nucleic acids have significant biological activities of their own that are deleterious to healthy cells of the body. Fragmented DNA and chromatin (DNAfs and Cfs) isolated from blood of cancer patients and healthy volunteers are readily taken up by a variety of cells in culture to be localized in their nuclei within a few minutes. The intra-nuclear DNAfs and Cfs associate themselves with host cell chromosomes to evoke a cellular DNA-damage-repair-response (DDR) followed by their incorporation into the host cell genomes. Whole genome sequencing detected the presence of tens of thousands of human sequence reads in the recipient mouse cells. Genomic incorporation of DNAfs and Cfs leads to dsDNA breaks and activation of apoptotic pathways in the treated cells. When injected intravenously into Balb/C mice, DNAfs and Cfs undergo genomic integration into cells of their vital organs resulting in activation of DDR and apoptotic proteins in the recipient cells. Cfs have significantly greater activity than DNAfs with respect to all parameters examined, while both DNAfs and Cfs isolated from cancer patients are more active than those from normal volunteers. All the above pathological actions of DNAfs and Cfs described above can be abrogated by concurrent treatment with DNase I and/or anti-histone antibody complexed nanoparticles both in vitro and in vivo. Taken together, our results that circulating DNAfs and Cfs are physiological, continuously arising, endogenous DNA damaging agents with implications to ageing and a multitude of human pathologies including initiation of cancer. PMID:25740145

  20. Reconstruction of damaged corneal epithelium using Venus-labeled limbal epithelial stem cells and tracking of surviving donor cells.

    PubMed

    Yin, Ji-Qing; Liu, Wen-Qiang; Liu, Chao; Zhang, Yi-Hua; Hua, Jin-Lian; Liu, Wei-Shuai; Dou, Zhong-Ying; Lei, An-Min

    2013-10-01

    Limbal epithelial stem cells are responsible for the self-renewal and replenishment of the corneal epithelium. Although it is possible to repair the ocular surface using limbal stem cell transplantation, the mechanisms behind this therapy are unclear. To investigate the distribution of surviving donor cells in a reconstructed corneal epithelium, we screened a Venus-labeled limbal stem cell strain in goats. Cells were cultivated on denuded human amniotic membrane for 21 days to produce Venus-labeled corneal epithelial sheets. The Venus-labeled corneal epithelial sheets were transplanted to goat models of limbal stem cell deficiency. At 3 months post-surgery, the damaged corneal epithelia were obviously improved in the transplanted group compared with the non-transplanted control, with the donor cells still residing in the reconstructed ocular surface epithelium. Using Venus as a marker, our results indicated that the location and survival of donor cells varied, depending on the corneal epithelial region. Additionally, immunofluorescent staining of the reconstructed corneal epithelium demonstrated that many P63(+) cells were unevenly distributed among basal and suprabasal epithelial layers. Our study provides a new model, and reveals some of the mechanisms involved in corneal epithelial cell regeneration research. Copyright © 2013 Elsevier Ltd. All rights reserved.

  1. Cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with accelerated 56Fe ions

    NASA Technical Reports Server (NTRS)

    Suzuki, M.; Piao, C.; Hall, E. J.; Hei, T. K.

    2001-01-01

    We examined cell killing and chromatid damage in primary human bronchial epithelial cells irradiated with high-energy 56Fe ions. Cells were irradiated with graded doses of 56Fe ions (1 GeV/nucleon) accelerated with the Alternating Gradient Synchrotron at Brookhaven National Laboratory. The survival curves for cells plated 1 h after irradiation (immediate plating) showed little or no shoulder. However, the survival curves for cells plated 24 h after irradiation (delayed plating) had a small initial shoulder. The RBE for 56Fe ions compared to 137Cs gamma rays was 1.99 for immediate plating and 2.73 for delayed plating at the D10. The repair ratio (delayed plating/immediate plating) was 1.67 for 137Cs gamma rays and 1.22 for 56Fe ions. The dose-response curves for initially measured and residual chromatid fragments detected by the Calyculin A-mediated premature chromosome condensation technique showed a linear response. The results indicated that the induction frequency for initially measured fragments was the same for 137Cs gamma rays and 56Fe ions. On the other hand, approximately 85% of the fragments induced by 137Cs gamma rays had rejoined after 24 h of postirradiation incubation; the corresponding amount for 56Fe ions was 37%. Furthermore, the frequency of chromatid exchanges induced by gamma rays measured 24 h after irradiation was higher than that induced by 56Fe ions. No difference in the amount of chromatid damage induced by the two types of radiations was detected when assayed 1 h after irradiation. The results suggest that high-energy 56Fe ions induce a higher frequency of complex, unrepairable damage at both the cellular and chromosomal levels than 137Cs gamma rays in the target cells for radiation-induced lung cancers.

  2. Damage of photoreceptor-derived cells in culture induced by light emitting diode-derived blue light

    PubMed Central

    Kuse, Yoshiki; Ogawa, Kenjiro; Tsuruma, Kazuhiro; Shimazawa, Masamitsu; Hara, Hideaki

    2014-01-01

    Our eyes are increasingly exposed to light from the emitting diode (LED) light of video display terminals (VDT) which contain much blue light. VDTs are equipped with televisions, personal computers, and smart phones. The present study aims to clarify the mechanism underlying blue LED light-induced photoreceptor cell damage. Murine cone photoreceptor-derived cells (661 W) were exposed to blue, white, or green LED light (0.38 mW/cm2). In the present study, blue LED light increased reactive oxygen species (ROS) production, altered the protein expression level, induced the aggregation of short-wavelength opsins (S-opsin), resulting in severe cell damage. While, blue LED light damaged the primary retinal cells and the damage was photoreceptor specific. N-Acetylcysteine (NAC), an antioxidant, protected against the cellular damage induced by blue LED light. Overall, the LED light induced cell damage was wavelength-, but not energy-dependent and may cause more severe retinal photoreceptor cell damage than the other LED light. PMID:24909301

  3. Effect of multikinase inhibitors on caspase-independent cell death and DNA damage in HER2-overexpressing breast cancer cells.

    PubMed

    Seoane, Samuel; Montero, Juan Carlos; Ocaña, Alberto; Pandiella, Atanasio

    2010-09-22

    The receptor tyrosine kinase, HER2, is overexpressed in approximately 25% of patients with breast cancer and is implicated in the aggressiveness of cancer. Targeting of HER2 signaling with trastuzumab, a monoclonal antibody that inhibits HER2 activity, has demonstrated clinical benefits. We investigated whether the antitumor activity of trastuzumab can be potentiated by dasatinib, a small-molecule tyrosine kinase inhibitor, on breast cancer cell lines that overexpress HER2 (BT474 and SKBR3) or have normal HER2 expression (MCF7 and T47D). Functional, biochemical, and gene expression microarray studies were performed to test the effect of trastuzumab, dasatinib, or a combination of trastuzumab and dasatinib on cell proliferation; HER activation; cell cycle; DNA damage; and apoptosis. The effect of drugs on mice (n = 6 per group) bearing xenograft tumors originating from HER2-overexpressing BT474 cells was assessed, and tumors were evaluated for an effect on volume, HER signaling, and DNA damage. All statistical tests were two-sided. Trastuzumab and dasatinib combination showed a synergistic effect on the proliferation of HER2-overexpressing breast cancer cells (combination index = 0.44, 95% confidence interval = 0.30 to 0.58). The drug combination also induced a stronger inhibitory effect on HER2 activation than the individual drugs, decreased the level of proteins involved in DNA damage response, induced DNA double-strand breaks, cell cycle arrest, and caspase-independent apoptosis. Mice (n = 6 per group) bearing xenograft tumors originating from HER2-overexpressing BT474 cells showed statistically significantly reduced tumor volume on day 28 when treated with the drug combination (control vs trastuzumab and dasatinib combination; mean volume = 2.6 vs 0.5 cm(3), difference = 2.1 cm(3), 95% confidence interval = 0.76 to 3.51 cm(3), P = .01) and total regression of tumors by day 36 with no later relapse. Results showed that HER2 and dasatinib-sensitive tyrosine

  4. Curcumin causes DNA damage and affects associated protein expression in HeLa human cervical cancer cells.

    PubMed

    Shang, Hung-Sheng; Chang, Chuan-Hsun; Chou, Yu-Ru; Yeh, Ming-Yang; Au, Man-Kuan; Lu, Hsu-Feng; Chu, Yung-Lin; Chou, Hsiao-Min; Chou, Hsiu-Chen; Shih, Yung-Luen; Chung, Jing-Gung

    2016-10-01

    Cervical cancer is one of the most common cancers in women worldwide and it is a prominent cause of cancer mortality. Curcumin is one of the major compounds from Turmeric and has been shown to induce cytotoxic cell death in human cervical cancer cells. However, there is no study to show curcumin induced DNA damage action via the effect on the DNA damage and repair protein in cervical cancer cells in detail. In this study, we investigated whether or not curcumin induced cell death via DNA damage, chromatin condensation in human cervical cancer HeLa cells by using comet assay and DAPI staining, respectively, we found that curcumin induced cell death through the induction of DNA damage, and chromatin condensation. Western blotting and confocal laser microscopy examination were used to examine the effects of curcumin on protein expression associated with DNA damage, repair and translocation of proteins. We found that curcumin at 13 µM increased the protein levels associated with DNA damage and repair, such as O6-methylguanine-DNA methyltransferase, early-onset breast cancer 1 (BRCA1), mediator of DNA damage checkpoint 1, p-p53 and p-H2A.XSer140 in HeLa cells. Results from confocal laser systems microscopy indicated that curcumin increased the translocation of p-p53 and p-H2A.XSer140 from cytosol to nuclei in HeLa cells. In conclusion, curcumin induced cell death in HeLa cells via induction of DNA damage, and chromatin condensation in vitro.

  5. FOXO3 Transcription Factor Is Essential for Protecting Hematopoietic Stem and Progenitor Cells from Oxidative DNA Damage.

    PubMed

    Bigarella, Carolina L; Li, Jianfeng; Rimmelé, Pauline; Liang, Raymond; Sobol, Robert W; Ghaffari, Saghi

    2017-02-17

    Accumulation of damaged DNA in hematopoietic stem cells (HSC) is associated with chromosomal abnormalities, genomic instability, and HSC aging and might promote hematological malignancies with age. Despite this, the regulatory pathways implicated in the HSC DNA damage response have not been fully elucidated. One of the sources of DNA damage is reactive oxygen species (ROS) generated by both exogenous and endogenous insults. Balancing ROS levels in HSC requires FOXO3, which is an essential transcription factor for HSC maintenance implicated in HSC aging. Elevated ROS levels result in defective Foxo3 -/- HSC cycling, among many other deficiencies. Here, we show that loss of FOXO3 leads to the accumulation of DNA damage in primitive hematopoietic stem and progenitor cells (HSPC), associated specifically with reduced expression of genes implicated in the repair of oxidative DNA damage. We provide further evidence that Foxo3 -/- HSPC are defective in DNA damage repair. Specifically, we show that the base excision repair pathway, the main pathway utilized for the repair of oxidative DNA damage, is compromised in Foxo3 -/- primitive hematopoietic cells. Treating mice in vivo with N -acetylcysteine reduces ROS levels, rescues HSC cycling defects, and partially mitigates HSPC DNA damage. These results indicate that DNA damage accrued as a result of elevated ROS in Foxo3 -/- mutant HSPC is at least partially reversible. Collectively, our findings suggest that FOXO3 serves as a protector of HSC genomic stability and health. © 2017 by The American Society for Biochemistry and Molecular Biology, Inc.

  6. E-Cigarette Aerosol Exposure Induces Reactive Oxygen Species, DNA Damage, and Cell Death in Vascular Endothelial Cells.

    PubMed

    Anderson, Chastain; Majeste, Andrew; Hanus, Jakub; Wang, Shusheng

    2016-12-01

    Cigarette smoking remains one of the leading causes of preventable death worldwide. Vascular cell death and dysfunction is a central or exacerbating component in the majority of cigarette smoking related pathologies. The recent development of the electronic nicotine delivery systems known as e-cigarettes provides an alternative to conventional cigarette smoking; however, the potential vascular health risks of e-cigarette use remain unclear. This study evaluates the effects of e-cigarette aerosol extract (EAE) and conventional cigarette smoke extract (CSE) on human umbilical vein endothelial cells (HUVECs). A laboratory apparatus was designed to produce extracts from e-cigarettes and conventional cigarettes according to established protocols for cigarette smoking. EAE or conventional CSE was applied to human vascular endothelial cells for 4-72 h, dependent on the assay. Treated cells were assayed for reactive oxygen species, DNA damage, cell viability, and markers of programmed cell death pathways. Additionally, the anti-oxidants α-tocopherol and n-acetyl-l-cysteine were used to attempt to rescue e-cigarette induced cell death. Our results indicate that e-cigarette aerosol is capable of inducing reactive oxygen species, causing DNA damage, and significantly reducing cell viability in a concentration dependent fashion. Immunofluorescent and flow cytometry analysis indicate that both the apoptosis and programmed necrosis pathways are triggered by e-cigarette aerosol treatment. Additionally, anti-oxidant treatment provides a partial rescue of the induced cell death, indicating that reactive oxygen species play a causal role in e-cigarette induced cytotoxicity. © The Author 2016. Published by Oxford University Press on behalf of the Society of Toxicology. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

  7. Characterization of DNA damage-dependent cell cycle checkpoints in a menin-deficient model.

    PubMed

    Kottemann, Molly C; Bale, Allen E

    2009-08-06

    MEN1, the gene responsible for the cancer predisposition syndrome multiple endocrine neoplasia type I, has been implicated in DNA repair, cell cycle control, and transcriptional regulation. It is unclear to what degree these processes are integrated into a single encompassing function in normal cellular physiology and how deficiency of the MEN1-encoded protein, "menin", contributes to cancer pathogenesis. In this study, we found that loss of Men1 in mouse embryonic fibroblasts caused abrogation of the G1/S and intra-S checkpoints following ionizing radiation. The cyclin-dependent kinase inhibitor, p21, failed to be upregulated in the mutant although upstream checkpoint signaling remained intact. Menin localized to the p21 promoter in a DNA damage-dependent manner. The MLL histone methyltransferase, a positive transcriptional regulator, bound to the same region in the presence of menin but not in Men1(-/-) cells. Finally, p53 retained damage-responsive binding to the p21 promoter in the Men1 mutant. These data indicate that menin participates in the checkpoint response in a transcriptional capacity, upregulating the DNA damage-responsive target p21.

  8. Effect of circadian clock mutations on DNA damage response in mammalian cells.

    PubMed

    Gaddameedhi, Shobhan; Reardon, Joyce T; Ye, Rui; Ozturk, Nuri; Sancar, Aziz

    2012-09-15

    The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents.

  9. Effect of circadian clock mutations on DNA damage response in mammalian cells

    PubMed Central

    Gaddameedhi, Shobhan; Reardon, Joyce T.; Ye, Rui; Ozturk, Nuri; Sancar, Aziz

    2012-01-01

    The circadian clock is a global regulatory mechanism that confers daily rhythmicity on many biochemical and physiological functions, including DNA excision repair in mammalian organisms. Here, we investigated the effect of the circadian clock on the major DNA damage response pathways by using mouse cell lines mutated in genes encoding proteins in the positive (Bmal1, CLOCK) or negative (Cry 1/2, Per 1/2) arms of the transcription-translation feedback loop that generates the circadian clock. We find that cells mutated in these genes are indistinguishable from wild-type in their response to UV, ionizing radiation and mitomycin C. We conclude that either the majority of DNA damage response reactions are not controlled by the circadian clock or that, even if such a control exists at the organism level, it is supplanted by homeostatic control mechanisms at the cellular level in tissue culture. We suggest that caution must be exercised in extrapolating from experiments in tissue culture to whole animals with respect to the effect of the circadian clock on cellular response to DNA damaging agents. PMID:22918252

  10. Antioxidant activity of Coriandrum sativum and protection against DNA damage and cancer cell migration.

    PubMed

    Tang, Esther L H; Rajarajeswaran, Jayakumar; Fung, Shin Yee; Kanthimathi, M S

    2013-12-09

    Coriandrum sativum is a popular culinary and medicinal herb of the Apiaceae family. Health promoting properties of this herb have been reported in pharmacognostical, phytochemical and pharmacological studies. However, studies on C. sativum have always focused on the aerial parts of the herb and scientific investigation on the root is limited. The aim of this research was to investigate the antioxidant and anticancer activities of C. sativum root, leaf and stem, including its effect on cancer cell migration, and its protection against DNA damage, with special focus on the roots. Powdered roots, leaves and stems of C. sativum were extracted through sequential extraction using hexane, dichloromethane, ethyl acetate, methanol and water. Total phenolic content, FRAP and DPPH radical scavenging activities were measured. Anti-proliferative activitiy on the breast cancer cell line, MCF-7, was assayed using the MTT assay. Activities of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, and of the caspases-3, -8 and -9 were assayed on treatment with the extract. Cell cycle progression was analysed using flow cytometry. The scratch motility assay was used to assess inhibition of MCF-7 cell migration. DNA damage in 3 T3-L1 fibroblasts was evaluated by the comet assay. The components in the extract were identified by HPLC and GC-MS. The ethyl acetate extract of C. sativum roots showed the highest antiproliferative activity on MCF-7 cells (IC50 = 200.0 ± 2.6 μg/mL) and had the highest phenolic content, FRAP and DPPH scavenging activities among the extracts. C. sativum root inhibited DNA damage and prevented MCF-7 cell migration induced by H2O2, suggesting its potential in cancer prevention and inhibition of metastasis. The extract exhibited anticancer activity in MCF-7 cells by affecting antioxidant enzymes possibly leading to H2O2 accumulation, cell cycle arrest at the G2/M phase and apoptotic cell death by the death receptor and

  11. Antioxidant activity of Coriandrum sativum and protection against DNA damage and cancer cell migration

    PubMed Central

    2013-01-01

    Background Coriandrum sativum is a popular culinary and medicinal herb of the Apiaceae family. Health promoting properties of this herb have been reported in pharmacognostical, phytochemical and pharmacological studies. However, studies on C. sativum have always focused on the aerial parts of the herb and scientific investigation on the root is limited. The aim of this research was to investigate the antioxidant and anticancer activities of C. sativum root, leaf and stem, including its effect on cancer cell migration, and its protection against DNA damage, with special focus on the roots. Methods Powdered roots, leaves and stems of C. sativum were extracted through sequential extraction using hexane, dichloromethane, ethyl acetate, methanol and water. Total phenolic content, FRAP and DPPH radical scavenging activities were measured. Anti-proliferative activitiy on the breast cancer cell line, MCF-7, was assayed using the MTT assay. Activities of the antioxidant enzymes, catalase, superoxide dismutase, glutathione peroxidase, and of the caspases-3, -8 and -9 were assayed on treatment with the extract. Cell cycle progression was analysed using flow cytometry. The scratch motility assay was used to assess inhibition of MCF-7 cell migration. DNA damage in 3 T3-L1 fibroblasts was evaluated by the comet assay. The components in the extract were identified by HPLC and GC-MS. Results The ethyl acetate extract of C. sativum roots showed the highest antiproliferative activity on MCF-7 cells (IC50 = 200.0 ± 2.6 μg/mL) and had the highest phenolic content, FRAP and DPPH scavenging activities among the extracts. C. sativum root inhibited DNA damage and prevented MCF-7 cell migration induced by H2O2, suggesting its potential in cancer prevention and inhibition of metastasis. The extract exhibited anticancer activity in MCF-7 cells by affecting antioxidant enzymes possibly leading to H2O2 accumulation, cell cycle arrest at the G2/M phase and apoptotic cell death by

  12. A Supramolecular Gel Approach to Minimize the Neural Cell Damage during Cryopreservation Process.

    PubMed

    Zeng, Jie; Yin, Yixia; Zhang, Li; Hu, Wanghui; Zhang, Chaocan; Chen, Wanyu

    2016-03-01

    The storage method for living cells is one of the major challenges in cell-based applications. Here, a novel supramolecular gel cryopreservation system (BDTC gel system) is introduced, which can observably increase the neural cell viability during cryopreservation process because this system can (1) confine the ice crystal growth in the porous of BDTC gel system, (2) decrease the amount of ice crystallization and cryopreservation system's freezing point, and (3) reduce the change rates of cell volumes and osmotic shock. In addition, thermoreversible BDTC supramolecular gel is easy to be removed after thawing so it does not hinder the adherence, growth, and proliferation of cells. The results of functionality assessments indicate that BDTC gel system can minimize the neural cell damage during cryopreservation process. This method will be potentially applied in cryopreservation of other cell types, tissues, or organs and will benefit cell therapy, tissue engineering, and organs transplantation. © 2015 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

  13. Mechanisms of DNA damage repair in adult stem cells and implications for cancer formation.

    PubMed

    Weeden, Clare E; Asselin-Labat, Marie-Liesse

    2018-01-01

    Maintenance of genomic integrity in tissue-specific stem cells is critical for tissue homeostasis and the prevention of deleterious diseases such as cancer. Stem cells are subject to DNA damage induced by endogenous replication mishaps or exposure to exogenous agents. The type of DNA lesion and the cell cycle stage will invoke different DNA repair mechanisms depending on the intrinsic DNA repair machinery of a cell. Inappropriate DNA repair in stem cells can lead to cell death, or to the formation and accumulation of genetic alterations that can be transmitted to daughter cells and so is linked to cancer formation. DNA mutational signatures that are associated with DNA repair deficiencies or exposure to carcinogenic agents have been described in cancer. Here we review the most recent findings on DNA repair pathways activated in epithelial tissue stem and progenitor cells and their implications for cancer mutational signatures. We discuss how deep knowledge of early molecular events leading to carcinogenesis provides insights into DNA repair mechanisms operating in tumours and how these could be exploited therapeutically. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. The oxygen-rich postnatal environment induces cardiomyocyte cell-cycle arrest through DNA damage response.

    PubMed

    Puente, Bao N; Kimura, Wataru; Muralidhar, Shalini A; Moon, Jesung; Amatruda, James F; Phelps, Kate L; Grinsfelder, David; Rothermel, Beverly A; Chen, Rui; Garcia, Joseph A; Santos, Celio X; Thet, SuWannee; Mori, Eiichiro; Kinter, Michael T; Rindler, Paul M; Zacchigna, Serena; Mukherjee, Shibani; Chen, David J; Mahmoud, Ahmed I; Giacca, Mauro; Rabinovitch, Peter S; Aroumougame, Asaithamby; Shah, Ajay M; Szweda, Luke I; Sadek, Hesham A

    2014-04-24

    The mammalian heart has a remarkable regenerative capacity for a short period of time after birth, after which the majority of cardiomyocytes permanently exit cell cycle. We sought to determine the primary postnatal event that results in cardiomyocyte cell-cycle arrest. We hypothesized that transition to the oxygen-rich postnatal environment is the upstream signal that results in cell-cycle arrest of cardiomyocytes. Here, we show that reactive oxygen species (ROS), oxidative DNA damage, and DNA damage response (DDR) markers significantly increase in the heart during the first postnatal week. Intriguingly, postnatal hypoxemia, ROS scavenging, or inhibition of DDR all prolong the postnatal proliferative window of cardiomyocytes, whereas hyperoxemia and ROS generators shorten it. These findings uncover a protective mechanism that mediates cardiomyocyte cell-cycle arrest in exchange for utilization of oxygen-dependent aerobic metabolism. Reduction of mitochondrial-dependent oxidative stress should be an important component of cardiomyocyte proliferation-based therapeutic approaches. Copyright © 2014 Elsevier Inc. All rights reserved.

  15. Alcohol metabolism in human cells causes DNA damage and activates the Fanconi anemia – breast cancer susceptibility (FA-BRCA) DNA damage response network

    PubMed Central

    Abraham, Jessy; Balbo, Silvia; Crabb, David; Brooks, P.J.

    2011-01-01

    Background We recently reported that exposure of human cells in vitro to acetaldehyde resulted in activation of the Fanconi anemia-breast cancer associated (FA-BRCA) DNA damage response network. Methods To determine whether intracellular generation of acetaldehyde from ethanol metabolism can cause DNA damage and activate the FA-BRCA network, we engineered HeLa cells to metabolize alcohol by expression of human alcohol dehydrogenase 1B. Results Incubation of HeLa-ADH1B cells with ethanol (20 mM) resulted in acetaldehyde accumulation in the media which was prevented by co-incubation with 4-methyl pyrazole (4-MP), a specific inhibitor of ADH. Ethanol treatment of HeLa-ADH1B cells produced a 4-fold increase in the acetaldehyde-DNA adduct, N2-ethylidene-dGuo, and also resulted in activation of the Fanconi anemia -breast cancer susceptibility (FA-BRCA) DNA damage response network, as indicated by a monoubiquitination of FANCD2, and phosphorylation of BRCA1. Ser 1524 was identified as one site of BRCA1 phosphorylation. The increased levels of DNA adducts, FANCD2 monoubiquitination, and BRCA1 phosphorylation were all blocked by 4-MP, indicating that acetaldehyde, rather than ethanol itself, was responsible for all three responses. Importantly, the ethanol concentration we used is within the range that can be attained in the human body during social drinking. Conclusions Our results indicate that intracellular metabolism of ethanol to acetaldehyde results in DNA damage which activates the FA-BRCA DNA damage response network. PMID:21919919

  16. Assessment of okadaic acid effects on cytotoxicity, DNA damage and DNA repair in human cells.

    PubMed

    Valdiglesias, Vanessa; Méndez, Josefina; Pásaro, Eduardo; Cemeli, Eduardo; Anderson, Diana; Laffon, Blanca

    2010-07-07

    Okadaic acid (OA) is a phycotoxin produced by several types of dinoflagellates causing diarrheic shellfish poisoning (DSP) in humans. Symptoms induced by DSP toxins are mainly gastrointestinal, but the intoxication does not appear to be fatal. Despite this, this toxin presents a potential threat to human health even at concentrations too low to induce acute toxicity, since previous animal studies have shown that OA has very potent tumour promoting activity. However, its concrete action mechanism has not been described yet and the results reported with regard to OA cytotoxicity and genotoxicity are often contradictory. In the present study, the genotoxic and cytotoxic effects of OA on three different types of human cells (peripheral blood leukocytes, HepG2 hepatoma cells, and SHSY5Y neuroblastoma cells) were evaluated. Cells were treated with a range of OA concentrations in the presence and absence of S9 fraction, and MTT test and Comet assay were performed in order to evaluate cytotoxicity and genotoxicity, respectively. The possible effects of OA on DNA repair were also studied by means of the DNA repair competence assay, using bleomycin as DNA damage inductor. Treatment with OA in absence of S9 fraction induced not statistically significant decrease in cell viability and significant increase in DNA damage in all cell types at the highest concentrations investigated. However, only SHSY5Y cells showed OA induced genotoxic and cytotoxic effects in presence of S9 fraction. Furthermore, we found that OA can induce modulations in DNA repair processes when exposure was performed prior to BLM treatment, in co-exposure, or during the subsequent DNA repair process. Copyright 2010 Elsevier B.V. All rights reserved.

  17. Protective role of NecroX-5 against neomycin-induced hair cell damage in zebrafish.

    PubMed

    Song, Jae-Jun; Chang, Jiwon; Choi, Jungim; Im, Gi Jung; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon-Young; Jung, Hak Hyun; Chung, Ah-Young; Park, Hae-Chul; Choi, June

    2014-02-01

    NecroX-5, one of the derivatives of NecroX series compounds, is a mitochondrial reactive oxygen species and reactive nitrogen species scavenger that inhibits cell death against various kinds of oxidative stresses. The objective of the present study was to evaluate the effects of NecroX-5 on neomycin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five days post-fertilization, zebrafish larvae were exposed to 125 μM neomycin and one of the following NecroX-5 concentrations for 1 h: 10, 25, 50, and 75 μM. Hair cells within the neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed using fluorescence microscopy (n = 10). The terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end labeling (TUNEL) assay and 2-[4-(dimethylamino) styryl]-N-ethylpyridiniumiodide (DASPEI) assay were performed for evaluation of apoptosis and mitochondrial damage. Ultrastructural changes were evaluated using scanning electron microscopy. NecroX-5 decreased neomycin-induced hair cell loss in the neuromasts (NecroX-5 50 μM: 13.4 ± 2.0 cells, 125 μM neomycin only: 8.1 ± 1.2 cells; n = 10, P < 0.05) and decreased the TUNEL reaction. The ultrastructural analysis showed that the structures of mitochondria and hair cells within the neuromasts were preserved in zebrafish exposed to 125 μM neomycin and 50 μM NecroX-5. NecroX-5 decreased apoptosis and mitochondrial damage. In conclusion, NecroX-5 attenuated neomycin-induced hair cell loss in zebrafish.

  18. Nitroglycerin induces DNA damage and vascular cell death in the setting of nitrate tolerance.

    PubMed

    Mikhed, Yuliya; Fahrer, Jörg; Oelze, Matthias; Kröller-Schön, Swenja; Steven, Sebastian; Welschof, Philipp; Zinßius, Elena; Stamm, Paul; Kashani, Fatemeh; Roohani, Siyer; Kress, Joana Melanie; Ullmann, Elisabeth; Tran, Lan P; Schulz, Eberhard; Epe, Bernd; Kaina, Bernd; Münzel, Thomas; Daiber, Andreas

    2016-07-01

    Nitroglycerin (GTN) and other organic nitrates are widely used vasodilators. Their side effects are development of nitrate tolerance and endothelial dysfunction. Given the potential of GTN to induce nitro-oxidative stress, we investigated the interaction between nitro-oxidative DNA damage and vascular dysfunction in experimental nitrate tolerance. Cultured endothelial hybridoma cells (EA.hy 926) and Wistar rats were treated with GTN (ex vivo: 10-1000 µM; in vivo: 10, 20 and 50 mg/kg/day for 3 days, s.c.). The level of DNA strand breaks, 8-oxoguanine and O (6)-methylguanine DNA adducts was determined by Comet assay, dot blot and immunohistochemistry. Vascular function was determined by isometric tension recording. DNA adducts and strand breaks were induced by GTN in cells in vitro in a concentration-dependent manner. GTN in vivo administration leads to endothelial dysfunction, nitrate tolerance, aortic and cardiac oxidative stress, formation of DNA adducts, stabilization of p53 and apoptotic death of vascular cells in a dose-dependent fashion. Mice lacking O (6)-methylguanine-DNA methyltransferase displayed more vascular O (6)-methylguanine adducts and oxidative stress under GTN therapy than wild-type mice. Although we were not able to prove a causal role of DNA damage in the etiology of nitrate tolerance, the finding of GTN-induced DNA damage such as the mutagenic and toxic adduct O (6)-methylguanine, and cell death supports the notion that GTN based therapy may provoke adverse side effects, including endothelial function. Further studies are warranted to clarify whether GTN pro-apoptotic effects are related to an impaired recovery of patients upon myocardial infarction.

  19. The pesticide Lindane induces dose-dependent damage to granulosa cells in an in vitro culture.

    PubMed

    Palmerini, Maria Grazia; Zhurabekova, Gulmira; Balmagambetova, Aru; Nottola, Stefania Annarita; Miglietta, Selenia; Belli, Manuel; Bianchi, Serena; Cecconi, Sandra; Di Nisio, Valentina; Familiari, Giuseppe; Macchiarelli, Guido

    2017-12-01

    Lindane, which is one of the most persistent organochlorine pesticide contaminating the Aral Sea region, is associated with numerous pathologies of the female reproductive system, including infertility, due to its gap junction blocker activity. By using an in vitro model of reproductive toxicity consisting of mouse parietal granulosa cells (GCs) exposed to increasing concentrations of Lindane ranging from 1 to 100μM (L1; L10; L100), we aimed to ascertain the Lindane toxicity by evaluating the ultrastructure and expression of the cell death protein p53. GCs exposed to L1 showed an early sign of degeneration as chromatin marginalization and initial reduction of cell-to-cell contacts. Such effects increased at L10 with nuclear membrane invagination, cytoplasmic blebbing, reduction of microvilli and intercellular connections. L100 induced evident cellular damages with an extensive presence of vacuoles, cytoplasmic fragments, nuclear membrane vesiculation and abundant cellular debris. A dose-dependent increase of p53 expression was evident in the L1 and L10 groups but not in L100. These data provide evidence for a dose-dependent reproductive toxicity of the gap junction blocker Lindane, as seen in mouse GCs cultured in vitro by ultrastructural damage compatible with apoptosis. Since gap junctions may play a critical role in FSH-stimulated progesterone production, the ultrastructural damage here evidenced could explain the increase in the prevalence of reproductive pathologies and infertility in exposed women. Finally, this study provided a useful and repeatable model of reproductive toxicity in vitro, which is applicable to evaluate the detrimental effects of toxicants or the reversing effect of protective substances. Copyright © 2017 Society for Biology of Reproduction & the Institute of Animal Reproduction and Food Research of Polish Academy of Sciences in Olsztyn. Published by Elsevier Urban & Partner Sp. z o.o. All rights reserved.

  20. Damage to the microbial cell membrane during pyrolytic sugar utilization and strategies for increasing resistance.

    PubMed

    Jin, Tao; Rover, Marjorie R; Petersen, Elspeth M; Chi, Zhanyou; Smith, Ryan G; Brown, Robert C; Wen, Zhiyou; Jarboe, Laura R

    2017-09-01

    Lignocellulosic biomass is an appealing feedstock for the production of biorenewable fuels and chemicals, and thermochemical processing is a promising method for depolymerizing it into sugars. However, trace compounds in this pyrolytic sugar syrup are inhibitory to microbial biocatalysts. This study demonstrates that hydrophobic inhibitors damage the cell membrane of ethanologenic Escherichia coli KO11+lgk. Adaptive evolution was employed to identify design strategies for improving pyrolytic sugar tolerance and utilization. Characterization of the resulting evolved strain indicates that increased resistance to the membrane-damaging effects of the pyrolytic sugars can be attributed to a glutamine to leucine mutation at position 29 of carbon storage regulator CsrA. This single amino acid change is sufficient for decreasing EPS protein production and increasing membrane integrity when exposed to pyrolytic sugars.

  1. Semiconductor structural damage attendant to contact formation in III-V solar cells

    NASA Technical Reports Server (NTRS)

    Fatemi, Navid S.; Weizer, Victor G.

    1991-01-01

    In order to keep the resistive losses in solar cells to a minimum, it is often necessary for the ohmic contacts to be heat treated to lower the metal-semiconductor contact resistivity to acceptable values. Sintering of the contacts, however can result in extensive mechanical damage of the semiconductor surface under the metallization. An investigation of the detailed mechanisms involved in the process of contact formation during heat treatment may control the structural damage incurred by the semiconductor surface to acceptable levels, while achieving the desired values of contact resistivity for the ohmic contacts. The reaction kinetics of sintered gold contacts to InP were determined. It was found that the Au-InP interaction involves three consecutive stages marked by distinct color changes observed on the surface of the Au, and that each stage is governed by a different mechanism. A detailed description of these mechanisms and options to control them are presented.

  2. High level methicillin resistance correlates with reduced Staphylococcus aureus endothelial cell damage.

    PubMed

    Seidl, Kati; Leemann, Michèle; Palheiros Marques, Miguel; Rachmühl, Carole; Leimer, Nadja; Andreoni, Federica; Achermann, Yvonne; Zinkernagel, Annelies S

    2017-01-01

    There has been controversy about the intrinsic virulence of methicillin-resistant Staphylococcus aureus (MRSA) as compared to methicillin-susceptible S. aureus (MSSA). To address this discrepancy, the intrinsic virulence of 42 MRSA and 40 MSSA clinical isolates was assessed by testing endothelial cell (EC) damage, a surrogate marker for virulence in blood stream infections. Since these clinical isolates represent a heterogeneous group, well characterized S. aureus laboratory strains with SCCmec loss- and gain-of-function mutations were used in addition. The clinical MRSA isolates carrying typical hospital acquired SCCmec types (I, II or III) induced significantly less damage (47.8%) as compared to isolates with other SCCmec types (62.3%, p=0.03) and MSSA isolates (64.2%, p<0.01). There was a strong inverse correlation between high-level oxacillin resistance and low EC damage induction (R 2 =0.4464, p<0.001). High-level oxacillin resistant strains (MIC >32μ/ml) grew significantly slower as compared to isolates with low-level resistance (p=0.047). The level of EC damage positively correlated with α- and δ-toxin production (p<0.0001 and p<0.05, respectively) but not with β-toxin production. Invasive MRSA isolates (n=21, 56.3%) were significantly less cytotoxic as compared to invasive MSSA isolates (n=20, 68.0%, p<0.05). There was no difference between EC damage induced by superficial versus invasive isolates in either MRSA or MSSA strains. Our data suggest that the intrinsic virulence of MRSA is similar or even reduced as compared to MSSA strains but is linked to the level of methicillin resistance. Copyright © 2016 Elsevier GmbH. All rights reserved.

  3. 3-Nitrobenzanthrone and 3-aminobenzanthrone induce DNA damage and cell signalling in Hepa1c1c7 cells.

    PubMed

    Landvik, N E; Arlt, V M; Nagy, E; Solhaug, A; Tekpli, X; Schmeiser, H H; Refsnes, M; Phillips, D H; Lagadic-Gossmann, D; Holme, J A

    2010-02-03

    3-Nitrobenzanthrone (3-NBA) is a mutagenic and carcinogenic environmental pollutant found in diesel exhaust and urban air pollution. In the present work we have characterised the effects of 3-NBA and its metabolite 3-aminobenzanthrone (3-ABA) on cell death and cytokine release in mouse hepatoma Hepa1c1c7 cells. These effects were related to induced DNA damage and changes in cell signalling pathways. 3-NBA resulted in cell death and caused most DNA damage as judged by the amount of DNA adducts ((32)P-postlabelling assay), single strand (ss)DNA breaks and oxidative DNA lesions (comet assay) detected. An increased phosphorylation of H2AX, chk1, chk2 and partly ATM was observed using flow cytometry and/or Western blotting. Both compounds increased phosphorylation of p53 and MAPKs (ERK, p38 and JNK). However, only 3-NBA caused an accumulation of p53 in the nucleus and a translocation of Bax to the mitochondria. The p53 inhibitor pifithrin-alpha inhibited 3-NBA-induced apoptosis, indicating that cell death was a result of the triggering of DNA signalling pathways. The highest phosphorylation of Akt and degradation of IkappaB-alpha (suggesting activation of NF-kappaB) were also seen after treatment with 3-NBA. In contrast 3-ABA increased IL-6 release, but caused little or no toxicity. Cytokine release was inhibited by PD98059 and curcumin, suggesting that ERK and NF-kappaB play a role in this process. In conclusion, 3-NBA seems to have a higher potency to induce DNA damage compatible with its cytotoxic effects, while 3-ABA seems to have a greater effect on the immune system. Copyright 2009 Elsevier B.V. All rights reserved.

  4. Radiofrequency radiation (900 MHz)-induced DNA damage and cell cycle arrest in testicular germ cells in swiss albino mice.

    PubMed

    Pandey, Neelam; Giri, Sarbani; Das, Samrat; Upadhaya, Puja

    2017-04-01

    Even though there are contradictory reports regarding the cellular and molecular changes induced by mobile phone emitted radiofrequency radiation (RFR), the possibility of any biological effect cannot be ruled out. In view of a widespread and extensive use of mobile phones, this study evaluates alterations in male germ cell transformation kinetics following RFR exposure and after recovery. Swiss albino mice were exposed to RFR (900 MHz) for 4 h and 8 h duration per day for 35 days. One group of animals was terminated after the exposure period, while others were kept for an additional 35 days post-exposure. RFR exposure caused depolarization of mitochondrial membranes resulting in destabilized cellular redox homeostasis. Statistically significant increases in the damage index in germ cells and sperm head defects were noted in RFR-exposed animals. Flow cytometric estimation of germ cell subtypes in mice testis revealed 2.5-fold increases in spermatogonial populations with significant decreases in spermatids. Almost fourfold reduction in spermatogonia to spermatid turnover (1C:2C) and three times reduction in primary spermatocyte to spermatid turnover (1C:4C) was found indicating arrest in the premeiotic stage of spermatogenesis, which resulted in loss of post-meiotic germ cells apparent from testis histology and low sperm count in RFR-exposed animals. Histological alterations such as sloughing of immature germ cells into the seminiferous tubule lumen, epithelium depletion and maturation arrest were also observed. However, all these changes showed recovery to varied degrees following the post-exposure period indicating that the adverse effects of RFR on mice germ cells are detrimental but reversible. To conclude, RFR exposure-induced oxidative stress causes DNA damage in germ cells, which alters cell cycle progression leading to low sperm count in mice.

  5. Epiblastin A Induces Reprogramming of Epiblast Stem Cells Into Embryonic Stem Cells by Inhibition of Casein Kinase 1.

    PubMed

    Ursu, Andrei; Illich, Damir J; Takemoto, Yasushi; Porfetye, Arthur T; Zhang, Miao; Brockmeyer, Andreas; Janning, Petra; Watanabe, Nobumoto; Osada, Hiroyuki; Vetter, Ingrid R; Ziegler, Slava; Schöler, Hans R; Waldmann, Herbert

    2016-04-21

    The discovery of novel small molecules that induce stem cell reprogramming and give efficient access to pluripotent stem cells is of major importance for potential therapeutic applications and may reveal novel insights into the factors controlling pluripotency. Chemical reprogramming of mouse epiblast stem cells (EpiSCs) into cells corresponding to embryonic stem cells (cESCs) is an inefficient process. In order to identify small molecules that promote this cellular transition, we analyzed the LOPAC library in a phenotypic screen monitoring Oct4-GFP expression and identified triamterene (TR) as initial hit. Synthesis of a TR-derived compound collection and investigation for reprogramming of EpiSCs into cESCs identified casein kinases 1 (CK1) α/δ/ɛ as responsible cellular targets of TR and unraveled the structural parameters that determine reprogramming. Delineation of a structure-activity relationship led to the development of Epiblastin A, which engages CK1 isoenzymes in cell lysate and induces efficient conversion of EpiSCs into cESCs. Copyright © 2016 Elsevier Ltd. All rights reserved.

  6. Organelle-based aggregation and retention of damaged proteins in asymmetrically dividing cells.

    PubMed

    Zhou, Chuankai; Slaughter, Brian D; Unruh, Jay R; Guo, Fengli; Yu, Zulin; Mickey, Kristen; Narkar, Akshay; Ross, Rhonda Trimble; McClain, Melainia; Li, Rong

    2014-10-23

    Aggregation of damaged or misfolded proteins is a protective mechanism against proteotoxic stress, abnormalities of which underlie many aging-related diseases. Here, we show that in asymmetrically dividing yeast cells, aggregation of cytosolic misfolded proteins does not occur spontaneously but requires new polypeptide synthesis and is restricted to the surface of ER, which harbors the majority of active translation sites. Protein aggregates formed on ER are frequently also associated with or are later captured by mitochondria, greatly constraining aggregate mobility. During mitosis, aggregates are tethered to well-anchored maternal mitochondria, whereas mitochondria acquired by the bud are largely free of aggregates. Disruption of aggregate-mitochondria association resulted in increased mobility and leakage of mother-accumulated aggregates into the bud. Cells with advanced replicative age exhibit gradual decline of aggregates-mitochondria association, likely contributing to their diminished ability to rejuvenate through asymmetric cell division.

  7. [Effects of calcium way of Schwann cells on damage of peripheral nerve induced by acrylamide].

    PubMed

    Xiao, Jingwei; Niu, Kailong; Meng, Huilin; Cui, Tao; Li, Zhongsheng; Li, Bin

    2009-11-01

    To explore the mechanism of Schwann cells on damage of peripheral nerve injured by acrylamide. (1) Animal model: the neurobehavior and pathologic changes within exposure and recovery period were observed. The changes of Synapsin-I and S-100beta were measured by immunohistochemical method. (2) Cell mode: in this study, primary culture of Schwann cells and dorsal root ganglion (DRG) neurons and the co-culture of the two cells were established before the test. The toxicity of ACR on nerve cells was measured by MTT. Intracellular calcium concentration was examined for the protection of Schwann cells. Toxic symptoms of female were occurred obviously at cumulative dose (300-400 mg/kg). The reparative changes were found significantly after recovering 4 to 5 weeks. The signals of S-100beta were weakened after exposure. But within the extended recovery period, its positive signals were gradually increased in consistent with the decrease of toxic symptoms. The changes of Synapsin-I were consistent with that of S-100beta. Compared with non co-cultured neurons, co-cultured neurons have significant lower intracellular calcium concentration injured by acrylamide. The calcium channel of Schwann cells effects on the repair of peripheral nerve which is injured by acrylamide and Synapsin-I may be a key role site.

  8. Lysergic acid diethylamide causes photoreceptor cell damage through inducing inflammatory response and oxidative stress.

    PubMed

    Hu, Qi-Di; Xu, Ling-Li; Gong, Yan; Wu, Guo-Hai; Wang, Yu-Wen; Wu, Shan-Jun; Zhang, Zhe; Mao, Wei; Zhou, Yu-Sheng; Li, Qin-Bo; Yuan, Jian-Shu

    2018-01-19

    Lysergic acid diethylamide (LSD), a classical hallucinogen, was used as a popular and notorious substance of abuse in various parts of the world. Its abuse could result in long-lasting abnormalities in retina and little is known about the exact mechanism. This study was to investigate the effect of LSD on macrophage activation state at non-toxic concentration and its resultant toxicity to photoreceptor cells. Results showed that cytotoxicity was caused by LSD on 661 W cells after co-culturing with RAW264.7 cells. Treatment with LSD-induced RAW264.7 cells to the M1 phenotype, releasing more pro-inflammatory cytokines, and increasing the M1-related gene expression. Moreover, after co-culturing with RAW264.7 cells, significant oxidative stress in 661 W cells treated with LSD was observed, by increasing the level of malondialdehyde (MDA) and reactive oxygen species (ROS), and decreasing the level of glutathione (GSH) and the activity of superoxide dismutase (SOD). Our study demonstrated that LSD caused photoreceptor cell damage by inducing inflammatory response and resultant oxidative stress, providing the scientific rationale for the toxicity of LSD to retina.

  9. The Milieu of Damaged Alveolar Epithelial Type 2 Cells Stimulates Alveolar Wound Repair by Endogenous and Exogenous Progenitors

    PubMed Central

    Buckley, Susan; Shi, Wei; Carraro, Gianni; Sedrakyan, Sargis; Da Sacco, Stefano; Driscoll, Barbara A.; Perin, Laura; De Filippo, Roger E.

    2011-01-01

    Alveolar epithelial integrity is dependent upon the alveolar milieu, yet the milieu of the damaged alveolar epithelial cell type 2 (AEC2) has been little studied. Characterization of its components may offer the potential for ex vivo manipulation of stem cells to optimize their therapeutic potential. We examined the cytokine profile of AEC2 damage milieu, hypothesizing that it would promote endogenous epithelial repair while recruiting cells from other locations and instructing their engraftment and differentiation. Bronchoalveolar lavage and lung extract from hyperoxic rats represented AEC2 in vivo damage milieu, and medium from a scratch-damaged AEC2 monolayer represented in vitro damage. CINC-2 and ICAM, the major cytokines detected by proteomic cytokine array in AEC2 damage milieu, were chemoattractive to normoxic AECs and expedited in vitro wound healing, which was blocked by their respective neutralizing antibodies. The AEC2 damage milieu was also chemotactic for exogenous uncommitted human amniotic fluid stem cells (hAFSCs), increasing migration greater than 20-fold. hAFSCs attached within an in vitro AEC2 wound and expedited wound repair by contributing cytokines migration inhibitory factor and plasminogen activator inhibitor 1 to the AEC2 damage milieu, which promoted wound healing. The AEC2 damage milieu also promoted differentiation of a subpopulation of hAFSCs to express SPC, TTF-1, and ABCA3, phenotypic markers of distal alveolar epithelium. Thus, the microenvironment created by AEC2 damage not only promotes autocrine repair but also can attract uncommitted stem cells, which further augment healing through cytokine secretion and differentiation. PMID:21700959

  10. The Protecting Effect of Deoxyschisandrin and Schisandrin B on HaCaT Cells against UVB-Induced Damage

    PubMed Central

    Hou, Wei; Gao, Wei; Wang, Datao; Liu, Qingxiu; Zheng, Siwen; Wang, Yingping

    2015-01-01

    Schisandra chinensis is a traditional Chinese medicine that has multiple biological activities, including antioxidant, anticancer, tonic, and anti-aging effects. Deoxyschisandrin (SA) and schisandrin B (SB), the two major lignans isolated from S. chinensis, exert high antioxidant activities in vitro and in vivo by scavenging free radicals, such as reactive oxygen species (ROS). Ultraviolet B-ray (UVB) radiation induces the production of ROS and DNA damage, which eventually leads to cell death by apoptosis. However, it is unknown whether SA or SB protects cells against UVB-induced cellular DNA damage. Our study showed that both SA and SB effectively protected HaCaT cells from UVB-induced cell death by antagonizing UVB-mediated production of ROS and induction of DNA damage. Our results showed that both SA and SB significantly prevented UVB-induced loss of cell viability using 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide (MTT) assays. Dichloro-dihydro-fluorescein diacetate (DCFH-DA) assays showed that the production of ROS following UVB exposure was inhibited by treatment with SA and SB. Moreover, SA and SB decreased the UVB-induced DNA damage in HaCaT cells by comet assays. In addition, SA and SB also prevented UVB-induced cell apoptosis and the cleavage of caspase-3, caspase-8 and caspase-9. In a word, our results imply that the antioxidants SA and SB could protect cells from UVB-induced cell damage via scavenging ROS. PMID:25978330

  11. A comprehensive model for the proliferation-quiescence decision in response to endogenous DNA damage in human cells.

    PubMed

    Heldt, Frank S; Barr, Alexis R; Cooper, Sam; Bakal, Chris; Novák, Béla

    2018-03-06

    Human cells that suffer mild DNA damage can enter a reversible state of growth arrest known as quiescence. This decision to temporarily exit the cell cycle is essential to prevent the propagation of mutations, and most cancer cells harbor defects in the underlying control system. Here we present a mechanistic mathematical model to study the proliferation-quiescence decision in nontransformed human cells. We show that two bistable switches, the restriction point (RP) and the G1/S transition, mediate this decision by integrating DNA damage and mitogen signals. In particular, our data suggest that the cyclin-dependent kinase inhibitor p21 (Cip1/Waf1), which is expressed in response to DNA damage, promotes quiescence by blocking positive feedback loops that facilitate G1 progression downstream of serum stimulation. Intriguingly, cells exploit bistability in the RP to convert graded p21 and mitogen signals into an all-or-nothing cell-cycle response. The same mechanism creates a window of opportunity where G1 cells that have passed the RP can revert to quiescence if exposed to DNA damage. We present experimental evidence that cells gradually lose this ability to revert to quiescence as they progress through G1 and that the onset of rapid p21 degradation at the G1/S transition prevents this response altogether, insulating S phase from mild, endogenous DNA damage. Thus, two bistable switches conspire in the early cell cycle to provide both sensitivity and robustness to external stimuli.

  12. Study characterizes how DNA-damaging anti-cancer drugs kill cancer cells | Center for Cancer Research

    Cancer.gov

    Patients whose cancer cells express the SLFN11 protein are more likely to respond to DNA-damaging anti-cancer drugs than those whose cancer cells don’t express SLFN11. In a new study, Center for Cancer Research investigators show how these drugs recruit SLFN11 to block replication and kill cancer cells. Read more…

  13. Role of transfused red blood cells for shock and coagulopathy within remote damage control resuscitation.

    PubMed

    Spinella, Philip C; Doctor, Allan

    2014-05-01

    The philosophy of damage control resuscitation (DCR) and remote damage control resuscitation (RDCR) can be summarized by stating that the goal is to prevent death from hemorrhagic shock by "staying out of trouble instead of getting out of trouble." In other words, it is preferred to arrest the progression of shock, rather than also having to reverse this condition after significant tissue damage and organ injury cascades are established. Moreover, to prevent death from exsanguination, a balanced approach to the treatment of both shock and coagulopathy is required. This was military doctrine during World War II, but seemed to be forgotten during the last half of the 20th century. Damage control resuscitation and RDCR have revitalized the approach, but there is still more to learn about the most effective and safe resuscitative strategies to simultaneously treat shock and hemorrhage. Current data suggest that our preconceived notions regarding the efficacy of standard issue red blood cells (RBCs) during the hours after transfusion may be false. Standard issue RBCs may not increase oxygen delivery and may in fact decrease it by disturbing control of regional blood flow distribution (impaired nitric oxide processing) and failing to release oxygen, even when perfusing hypoxic tissue (abnormal oxygen affinity). Standard issue RBCs may assist with hemostasis but appear to have competing effects on thrombin generation and platelet function. If standard issue or RBCs of increased storage age are not optimal, then are there alternatives that will allow for an efficacious and safe treatment of shock while also supporting hemostasis? Studies are required to determine if fresh RBCs less than 7 to 10 days provide an outcome advantage. A resurgence in the study of whole blood stored at 4°C for up to 10 days also holds promise. Two randomized controlled trials in humans have indicated that following transfusion with either whole blood stored at 4°C or platelets stored at 4

  14. Gamma irradiation of the fetus damages the developing hemopoietic microenvironment rather than the hemopoietic progenitor cells

    SciTech Connect

    Yang, F.T.; Lord, B.I.; Hendry, J.H.

    1995-03-01

    Hemopoiesis is the product of two components: the hemopoietic tissue and the regulatory stromal microenvironment in which it resides. Plutonium-239, incorporated during fetal development in mice, is known to cause deficient hemopoiesis. A predetermined equivalent {gamma}-ray dose has now been used in combination with cross-transplantation experiments to separate these two components and define where the damage arises. It was confirmed that 1.8 Gy {gamma} irradiation at midterm gestation caused a 40% reduction in the hemopoietic stem (spleen colony-forming) cell population of their offspring which persisted to at least 24 weeks of age. Spleen colony formation after sublethal doses of {gamma}more » rays reflected this reduced complement of endogenous stem cells. The regulatory hemopoietic microenvironment, measured as fibroblastoid colony-forming cells, was similarly depleted. Normal growth of the CFU-S population after transplantation into standard recipients showed that the quality of the stem cell population in the offspring of irradiated mothers was not affected. By contrast, when used as recipients of a bone marrow transplant from either normal or irradiated offspring, the offspring of irradiated mothers were unable to support normal growth: there was a twofold difference in the number of CFU-S per femur for at least 100 days after transplantation. There were 70% fewer CFU-F in the femur 1 month after bone marrow transplantation when the offspring of irradiated mothers were used as transplant recipients compared to when normal offspring were used. This not only confirmed their reduced capacity to host normal stem cells but also indicated that CFU-F in the transplant were unable to compensate for the poor microenvironment in the irradiated offspring hosts. It is concluded that irradiation at midterm gestation damages the developing regulatory microenvironment but not the hemopoietic stem cell population that it hosts. 12 refs., 1 fig., 4 tabs.« less

  15. Acrolein preferentially damages nucleolus eliciting ribosomal stress and apoptosis in human cancer cells.

    PubMed

    Wang, Hsiang-Tsui; Chen, Tzu-Ying; Weng, Ching-Wen; Yang, Chun-Hsiang; Tang, Moon-Shong

    2016-12-06

    Acrolein (Acr) is a potent cytotoxic and DNA damaging agent which is ubiquitous in the environment and abundant in tobacco smoke. Acr is also an active cytotoxic metabolite of the anti-cancer drugs cyclophosphamide and ifosfamide. The mechanisms via which Acr exerts its anti-cancer activity and cytotoxicity are not clear. In this study, we found that Acr induces cytotoxicity and cell death in human cancer cells with different activities of p53. Acr preferentially binds nucleolar ribosomal DNA (rDNA) to form Acr-deoxyguanosine adducts, and induces oxidative damage to both rDNA and ribosomal RNA (rRNA). Acr triggers ribosomal stress responses, inhibits rRNA synthesis, reduces RNA polymerase I binding to the promoter of rRNA gene, disrupts nucleolar integrity, and impairs ribosome biogenesis and polysome formation. Acr causes an increase in MDM2 levels and phosphorylation of MDM2 in A549 and HeLa cells which are p53 active and p53 inactive, respectively. It enhances the binding of ribosomal protein RPL11 to MDM2 and reduces the binding of p53 and E2F-1 to MDM2 resulting in stabilization/activation of p53 in A549 cells and degradation of E2F-1 in A549 and HeLa cells. We propose that Acr induces ribosomal stress which leads to activation of MDM2 and RPL11-MDM2 binding, consequently, activates p53 and enhances E2F-1 degradation, and that taken together these two processes induce apoptosis and cell death.

  16. Indoleamine 2,3-dioxygenase protects corneal endothelial cells from UV mediated damage.

    PubMed

    Serbecic, Nermin; Beutelspacher, Sven Christoph

    2006-03-01

    Indoleamine-2,3-dioxygenase (IDO) is an intracellular enzyme present in dendritic cells and macrophages. It is a known modulator of T-cell response and contributes to the UV protection of the lens. There yet is no information on IDO activity in the corneal endothelium, protecting the endothelial cells from light mediated damage. We exposed murine corneal endothelial cells (MCEC) with different doses of UV-B light 280-320 nm, probed for IDO mRNA (real-time PCR) and assessed apoptosis rate (flow cytometry) and caspase-3-activity in the cells. The metabolites of the IDO catalysed reaction, l-kynurenine, was also measured. Malondialdehyde was detected for quantification of UV-B-induced oxidative stress. To investigate specificity, IDO effects were blocked by 1-methyl-tryptophan. The effects of IDO overexpression in the MCEC were assessed by transfection of an expression vector. MCEC consistently express IDO at low levels. Exposure to UV-B light led to a dose-responding upregulation of IDO; IDO was found competent converting l-tryptophan into l-kynurenine. Irradiation led to increased apoptosis and caspase-3-activity of MCEC. Supplementation of l-kynurenine or overexpression of IDO in the MCEC could reduce apoptosis significantly following UV-B irradiation. Inhibition of IDO by 1-MT was potent to reverse this effect. IDO and its metabolite l-kynurenine can protect corneal endothelial cells from UV-B-induced oxidative stress and apoptosis. It may be an active protection mechanism against corneal endothelial damage.

  17. Puerarin may protect against Schwann cell damage induced by glucose fluctuation.

    PubMed

    Xue, Bing; Wang, Lin; Zhang, Zhe; Wang, Rui; Xia, Xin-Xin; Han, Ping-Ping; Cao, Li-Jun; Liu, Yong-Hui; Sun, Lian-Qing

    2017-07-01

    Puerarin is one of the major active ingredients in Gegen, a traditional Chinese herb that has been reported to have a wide variety of beneficial pharmacology functions. Previous studies have implicated that the damaging effects of hyperglycemia resulting from oxidative stress and glucose fluctuation may be more dangerous than constant high glucose in the development of diabetes-related complications. The present study focuses on the effects of puerarin on glucose fluctuation-induced oxidative stress-induced Schwann cell (SC) apoptosis in vitro. Primarily cultured SCs were exposed to different conditions and the effect of puerarin on cell viability was determined by MTT assays. Intracellular reactive oxygen species (ROS) generation and mitochondrial transmembrane potential were detected by flow cytometry analysis. Apoptosis was confirmed by the Annexin V-FITC/PI and TUNEL method. Quantitative real-time reverse transcriptase polymerase chain reaction was performed to analyze the expression levels of bax and bcl-2. Western blot was performed to analyze the expression levels of some important transcription factors and proteins. The results showed that incubating SCs with intermittent high glucose for 48 h decreased cell viability and increased the number of apoptotic cells whereas treating with puerarin protected SCs against glucose fluctuation-induced cell damage. Further study demonstrated that puerarin suppressed activation of apoptosis-related proteins including PARP and caspase-3, downregulation of bcl-2, and upregulation of intracellular distribution of bax from cytosol to mitochondria, which was induced by glucose fluctuation. Moreover, puerarin inhibited the elevation of intracellular ROS and mitochondrial depolarization induced by glucose fluctuation. These results suggest that puerarin may protect SCs against glucose fluctuation-induced cell injury through inhibiting apoptosis as well as oxidative stress.

  18. Class III bacteriocin Helveticin-M causes sublethal damage on target cells through impairment of cell wall and membrane.

    PubMed

    Sun, Zhilan; Wang, Xiaomeng; Zhang, Xinxiao; Wu, Haihong; Zou, Ye; Li, Pengpeng; Sun, Chong; Xu, Weimin; Liu, Fang; Wang, Daoying

    2018-03-01

    Helveticin-M, a novel Class III bacteriocin produced by Lactobacillus crispatus exhibited an antimicrobial activity against Staphylococcus aureus, S. saprophyticus, and Enterobacter cloacae. To understand how Helveticin-M injured target cells, Helveticin-M was cloned and heterologously expressed in Escherichia coli. Subsequently, the cell wall organization and cell membrane integrity of target cells were determined. The mechanism of cellular damage differed according to bacterial species. Based on morphology analysis, Helveticin-M disrupted the cell wall of Gram-positive bacteria and disorganized the outer membrane of Gram-negative bacteria, therefore, altering surface structure. Helveticin-M also disrupted the inner membrane, as confirmed by leakage of intracellular ATP from cells and depolarization of membrane potential of target bacteria. Based on cell population analysis, Helveticin-M treatment caused the increase of cell membrane permeability, but the cytosolic enzymes were not influenced, indicating that it was the sublethal injury. Therefore, the mode of Helveticin-M action is bacteriostatic rather than bactericidal.

  19. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage

    PubMed Central

    Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

    2016-01-01

    Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis. PMID:27433029

  20. Effects of Chinese Propolis in Protecting Bovine Mammary Epithelial Cells against Mastitis Pathogens-Induced Cell Damage.

    PubMed

    Wang, Kai; Jin, Xiao-Lu; Shen, Xiao-Ge; Sun, Li-Ping; Wu, Li-Ming; Wei, Jiang-Qin; Marcucci, Maria Cristina; Hu, Fu-Liang; Liu, Jian-Xin

    2016-01-01

    Chinese propolis (CP), an important hive product, can alleviate inflammatory responses. However, little is known regarding the potential of propolis treatment for mastitis control. To investigate the anti-inflammatory effects of CP on bovine mammary epithelial cells (MAC-T), we used a range of pathogens to induce cellular inflammatory damage. Cell viability was determined and expressions of inflammatory/antioxidant genes were measured. Using a cell-based reporter assay system, we evaluated CP and its primary constituents on the NF-κB and Nrf2-ARE transcription activation. MAC-T cells treated with bacterial endotoxin (lipopolysaccharide, LPS), heat-inactivated Escherichia coli, and Staphylococcus aureus exhibited significant decreases in cell viability while TNF-α and lipoteichoic acid (LTA) did not. Pretreatment with CP prevented losses in cell viability associated with the addition of killed bacteria or bacterial endotoxins. There were also corresponding decreases in expressions of proinflammatory IL-6 and TNF-α mRNA. Compared with the mastitis challenged cells, enhanced expressions of antioxidant genes HO-1, Txnrd-1, and GCLM were observed in CP-treated cells. CP and its polyphenolic active components (primarily caffeic acid phenethyl ester and quercetin) had strong inhibitive effects against NF-κB activation and increased the transcriptional activity of Nrf2-ARE. These findings suggest that propolis may be valuable in the control of bovine mastitis.

  1. DNA-damage response network at the crossroads of cell-cycle checkpoints, cellular senescence and apoptosis.

    PubMed

    Schmitt, Estelle; Paquet, Claudie; Beauchemin, Myriam; Bertrand, Richard

    2007-06-01

    Tissue homeostasis requires a carefully-orchestrated balance between cell proliferation, cellular senescence and cell death. Cells proliferate through a cell cycle that is tightly regulated by cyclin-dependent kinase activities. Cellular senescence is a safeguard program limiting the proliferative competence of cells in living organisms. Apoptosis eliminates unwanted cells by the coordinated activity of gene products that regulate and effect cell death. The intimate link between the cell cycle, cellular senescence, apoptosis regulation, cancer development and tumor responses to cancer treatment has become eminently apparent. Extensive research on tumor suppressor genes, oncogenes, the cell cycle and apoptosis regulatory genes has revealed how the DNA damage-sensing and -signaling pathways, referred to as the DNA-damage response network, are tied to cell proliferation, cell-cycle arrest, cellular senescence and apoptosis. DNA-damage responses are complex, involving "sensor" proteins that sense the damage, and transmit signals to "transducer" proteins, which, in turn, convey the signals to numerous "effector" proteins implicated in specific cellular pathways, including DNA repair mechanisms, cell-cycle checkpoints, cellular senescence and apoptosis. The Bcl-2 family of proteins stands among the most crucial regulators of apoptosis and performs vital functions in deciding whether a cell will live or die after cancer chemotherapy and irradiation. In addition, several studies have now revealed that members of the Bcl-2 family also interface with the cell cycle, DNA repair/recombination and cellular senescence, effects that are generally distinct from their function in apoptosis. In this review, we report progress in understanding the molecular networks that regulate cell-cycle checkpoints, cellular senescence and apoptosis after DNA damage, and discuss the influence of some Bcl-2 family members on cell-cycle checkpoint regulation.

  2. Cationic Organochalcogen with Monomer/Excimer Emissions for Dual-Color Live Cell Imaging and Cell Damage Diagnosis.

    PubMed

    Chao, Xi-Juan; Wang, Kang-Nan; Sun, Li-Li; Cao, Qian; Ke, Zhuo-Feng; Cao, Du-Xia; Mao, Zong-Wan

    2018-04-25

    Studies on the development of fluorescent organic molecules with different emission colors for imaging of organelles and their biomedical application are gaining lots of focus recently. Here, we report two cationic organochalcogens 1 and 2, both of which exhibit very weak green emission (Φ 1 = 0.12%; Φ 2 = 0.09%) in dilute solution as monomers, but remarkably enhanced green emission upon interaction with nucleic acids and large red-shifted emission in aggregate state by the formation of excimers at high concentration. More interestingly, the monomer emission and excimer-like emission can be used for dual color imaging of different organelles. Upon passively diffusing into cells, both probes selectively stain nucleoli with strong green emission upon 488 nm excitation, whereas upon 405 nm excitation, a completely different stain pattern by staining lysosomes (for 1) or mitochondria (for 2) with distinct red emission is observed because of the highly concentrated accumulation in these organelles. Studies on the mechanism of the accumulation in lysosomes (for 1) or mitochondria (for 2) found that the accumulations of the probes are dependent on the membrane permeabilization, which make the probes have great potential in diagnosing cell damage by sensing lysosomal or mitochondrial membrane permeabilization. The study is demonstrative, for the first time, of two cationic molecules for dual-color imaging nucleoli and lysosomes (1)/mitochondria (2) simultaneously in live cell based on monomer and excimer-like emission, respectively, and more importantly, for diagnosing cell damage.

  3. DNA damage and DNA damage response in human bronchial epithelial BEAS-2B cells following exposure to 2-nitrobenzanthrone and 3-nitrobenzanthrone: role in apoptosis.

    PubMed

    Oya, Elisabeth; Ovrevik, Johan; Arlt, Volker M; Nagy, Eszter; Phillips, David H; Holme, Jørn A

    2011-11-01

    Nitro-polycyclic aromatic hydrocarbons (nitro-PAHs) are mutagenic and carcinogenic environmental pollutants found in diesel exhaust and on urban air pollution particles. In the present study, human bronchial epithelial BEAS-2B cells were exposed to 2-nitrobenzanthrone (2-NBA) and 3-nitrobenzanthrone (3-NBA). DNA damage responses were compared to those observed after exposure to 1-nitropyrene (1-NP) and benzo[a]pyrene (B[a]P). Examination by microscopy revealed that 3-NBA was the most potent toxic compound while weaker responses were observed with 1-NP and B[a]P. Most interestingly, 2-NBA did not induce cell death or any other stress-related responses. 3-NBA induced a typical apoptotic cell death judged by nuclear condensation and little plasma membrane damage as well as cleavage of caspase 3 and poly-(ADP-ribose) polymerase (PARP). Exposure to 3-NBA resulted in an accumulation of cells in S-phase, and further analysis by Western blotting, immunocytochemistry and flow cytometry revealed that 3-NBA induced a DNA damage response characterized by phosphorylation of ATM (ataxia-telangiectasia mutated), checkpoint kinase (Chk) 2/Chk1, H2AX and p53. The p53 inhibitor pifithrin-α inhibited 3-NBA-induced apoptosis while small effects were seen using pifithrin-μ, suggesting that 3-NBA-induced cell death is a result of transcriptional activation of p53. In conclusion, 3-NBA is a potent inducer of apoptosis, which seemed to be triggered by the DNA damage response. Furthermore, a change of the nitro-group to the second position (i.e. 2-NBA) dramatically changed the cellular reactivity of the compound.

  4. Clustered DNA damages induced in isolated DNA and in human cells by low doses of ionizing radiation

    NASA Technical Reports Server (NTRS)

    Sutherland, B. M.; Bennett, P. V.; Sidorkina, O.; Laval, J.; Lowenstein, D. I. (Principal Investigator)

    2000-01-01

    Clustered DNA damages-two or more closely spaced damages (strand breaks, abasic sites, or oxidized bases) on opposing strands-are suspects as critical lesions producing lethal and mutagenic effects of ionizing radiation. However, as a result of the lack of methods for measuring damage clusters induced by ionizing radiation in genomic DNA, neither the frequencies of their production by physiological doses of radiation, nor their repairability, nor their biological effects are known. On the basis of methods that we developed for quantitating damages in large DNAs, we have devised and validated a way of measuring ionizing radiation-induced clustered lesions in genomic DNA, including DNA from human cells. DNA is treated with an endonuclease that induces a single-strand cleavage at an oxidized base or abasic site. If there are two closely spaced damages on opposing strands, such cleavage will reduce the size of the DNA on a nondenaturing gel. We show that ionizing radiation does induce clustered DNA damages containing abasic sites, oxidized purines, or oxidized pyrimidines. Further, the frequency of each of these cluster classes is comparable to that of frank double-strand breaks; among all complex damages induced by ionizing radiation, double-strand breaks are only about 20%, with other clustered damage constituting some 80%. We also show that even low doses (0.1-1 Gy) of high linear energy transfer ionizing radiation induce clustered damages in human cells.

  5. The ACE2-angiotensin-(1-7)-Mas axis protects against pancreatic cell damage in cell culture.

    PubMed

    Wang, Jing; Liu, Ruixia; Qi, Haiyu; Wang, Yan; Cui, Lijian; Wen, Yan; Li, Huihui; Yin, Chenghong

    2015-03-01

    Angiotensin-converting enzyme 2 (ACE2), its product angiotensin-(1-7), and its receptor Mas have been shown to moderate the adverse effects of the ACE-angiotensin II-AT1 axis in many diseases. The aim of this study was to determine whether the ACE2-Ang-(1-7)-Mas axis could have similar effects in a cell culture model of pancreatic damage. AR42J cells were stimulated with 10 nmol/L cerulein to simulate acute pancreatitis. ACE2, Ang-(1-7), Mas receptor, and PI3K/AKT pathway were measured by quantitative real-time polymerase chain reaction and Western blot analysis. ACE2 and Mas receptor protein levels in AR42J cells were significantly increased (P < 0.05) between 30 minutes and 6 hours postdisease induction compared with the control group. Mas receptor gene expression was significantly increased (P < 0.05) at 2 hours postdisease induction, and Ang-(1-7) was increased at 6 hours. Treatment with Ang-(1-7) in AR42J cells increased IL-10, decreased IL-6 and IL-8, and reduced the damage to pancreatic cells. Levels of IL-6 and IL-8 in AR42J cell culture were increased significantly after treatment with A779. Moreover, Ang-(1-7) increased the concentration of PI3K/AKT pathway and eNOSin AR42J cells. ACE2-angiotensin-(1-7)-Mas axis significantly inhibits pancreatitis in response to decreased inflammatory factors by the activation of endothelial nitric oxide synthase and NO signaling pathways.

  6. Mangiferin aglycone attenuates radiation-induced damage on human intestinal epithelial cells.

    PubMed

    Lei, Jixiao; Zhou, Chuanfeng; Hu, Honggang; Hu, Lina; Zhao, Meijia; Yang, Yanyong; Chuai, Yunhai; Ni, Jin; Cai, Jianming

    2012-08-01

    Recent studies suggest that mangiferin aglycone (norathyriol) has great potential as a novel radioprotector without any known toxic side effects. In this study, we assessed the protective effects of mangiferin aglycone against radiation-induced injuries on normal human intestinal epithelial cells (HIECs), while using mangiferin as a reference compound. The in vitro experiments showed that pretreatment of either mangiferin aglycone or mangiferin could inhibit cytotoxic effects of ionizing irradiation (IR) on HIECs. Cellular changes were estimated by measuring cell viability, clonogenic surviving rate, and apoptotic rate. Compared to mangiferin, we found mangiferin aglycone had greater radioprotective effects of mangiferin aglycone on HIECs. It has been demonstrated that the cytotoxicity of ionizing radiation relates to its capacity to induce DNA damage. In view of this, we monitored DNA double-strand breaks (DSBs) using γH2AX foci formation to test whether mangiferin aglycone and mangiferin could modulate genotoxic effects of radiation. It shows that mangiferin aglycone could eliminate 46.8% of the total DSBs of the cells exposed to 2 Gy IR, which is significantly better than mangiferin. Complementing earlier results from our group, it appears possible to conclude that mangiferin aglycone presents potential useful effects on IR-induced damage and may be a better radioprotective agent than mangiferin therapeutically. Copyright © 2012 Wiley Periodicals, Inc.

  7. Oxidative stress-induced CREB upregulation promotes DNA damage repair prior to neuronal cell death protection.

    PubMed

    Pregi, Nicolás; Belluscio, Laura María; Berardino, Bruno Gabriel; Castillo, Daniela Susana; Cánepa, Eduardo Tomás

    2017-01-01

    cAMP response element-binding (CREB) protein is a cellular transcription factor that mediates responses to different physiological and pathological signals. Using a model of human neuronal cells we demonstrate herein, that CREB is phosphorylated after oxidative stress induced by hydrogen peroxide. This phosphorylation is largely independent of PKA and of the canonical phosphoacceptor site at ser-133, and is accompanied by an upregulation of CREB expression at both mRNA and protein levels. In accordance with previous data, we show that CREB upregulation promotes cell survival and that its silencing results in an increment of apoptosis after oxidative stress. Interestingly, we also found that CREB promotes DNA repair after treatment with hydrogen peroxide. Using a cDNA microarray we found that CREB is responsible for the regulation of many genes involved in DNA repair and cell survival after oxidative injury. In summary, the neuroprotective effect mediated by CREB appears to follow three essential steps following oxidative injury. First, the upregulation of CREB expression that allows sufficient level of activated and phosphorylated protein is the primordial event that promotes the induction of genes of the DNA Damage Response. Then and when the DNA repair is effective, CREB induces detoxification and survival genes. This kinetics seems to be important to completely resolve oxidative-induced neuronal damages.

  8. Dual protection of hydroxytyrosol, an olive oil polyphenol, against oxidative damage in PC12 cells.

    PubMed

    Peng, Shoujiao; Zhang, Baoxin; Yao, Juan; Duan, Dongzhu; Fang, Jianguo

    2015-06-01

    Hydroxytyrosol (3,4-dihydroxyphenylethanol, HT), a major polyphenol in olive oils, has received increasing attention due to its multiple pharmacological activities. However, it is not well understood how HT works on the neuronal system. We report herein that HT efficiently scavenges free radicals in vitro and displays cytoprotection against oxidative stress-induced damage in PC12 cells. HT completely protects the cells from hydrogen peroxide-induced death and rescues the cells from 6-hydroxydopamine-induced damage. Mechanistic studies reveal that Nrf2 is a prerequisite for the neuroprotection of HT as knocking down Nrf2 eliminated this action. HT, via activation of the Keap1-Nrf2 pathway, elevates a panel of cytoprotective enzymes, including glutamate-cysteine ligase, HO-1, NQO1 and thioredoxin reductase. Our study reveals that HT provides dual neuroprotection and cellular antioxidant defense as both a free radical scavenger and Nrf2 activator, suggesting the potential pharmaceutical usage of HT for the treatment of neurodegenerative disorders.

  9. Cyclin D1 depletion induces DNA damage in mantle cell lymphoma lines.

    PubMed

    Mohanty, Suchismita; Mohanty, Atish; Sandoval, Natalie; Tran, Thai; Bedell, Victoria; Wu, Jun; Scuto, Anna; Murata-Collins, Joyce; Weisenburger, Dennis D; Ngo, Vu N

    2017-03-01

    Elevated cyclin D1 (CCND1) expression levels in mantle cell lymphoma (MCL) are associated with aggressive clinical manifestations related to chemoresistance, but little is known about how this important proto-oncogene contributes to the resistance of MCL. Here, we showed that RNA interference-mediated depletion of CCND1 increased caspase-3 activities and induced apoptosis in the human MCL lines UPN-1 and JEKO-1. In vitro and xenotransplant studies revealed that the toxic effect of CCND1 depletion in MCL cells was likely due to increase in histone H2AX phosphorylation, a DNA damage marker. DNA fiber analysis suggested deregulated replication initiation after CCND1 depletion as a potential cause of DNA damage. Finally, in contrast to depletion or inhibition of cyclin-dependent kinase 4, CCND1 depletion increased chemosensitivity of MCL cells to replication inhibitors hydroxyurea and cytarabine. Our findings have an important implication for CCND1 as a potential therapeutic target in MCL patients who are refractory to standard chemotherapy.

  10. Persistent DNA Damage in Spermatogonial Stem Cells After Fractionated Low-Dose Irradiation of Testicular Tissue

    SciTech Connect

    Grewenig, Angelika; Schuler, Nadine; Rübe, Claudia E., E-mail: claudia.ruebe@uks.eu

    2015-08-01

    Purpose: Testicular spermatogenesis is extremely sensitive to radiation-induced damage, and even low scattered doses to testis from radiation therapy may pose reproductive risks with potential treatment-related infertility. Radiation-induced DNA double-strand breaks (DSBs) represent the greatest threat to the genomic integrity of spermatogonial stem cells (SSCs), which are essential to maintain spermatogenesis and prevent reproduction failure. Methods and Materials: During daily low-dose radiation with 100 mGy or 10 mGy, radiation-induced DSBs were monitored in mouse testis by quantifying 53 binding protein 1 (53BP-1) foci in SSCs within their stem cell niche. The accumulation of DSBs was correlated with proliferation, differentiation, and apoptosis ofmore » testicular germ cell populations. Results: Even very low doses of ionizing radiation arrested spermatogenesis, primarily by inducing apoptosis in spermatogonia. Eventual recovery of spermatogenesis depended on the survival of SSCs and their functional ability to proliferate and differentiate to provide adequate numbers of differentiating spermatogonia. Importantly, apoptosis-resistant SSCs resulted in increased 53BP-1 foci levels during, and even several months after, fractionated low-dose radiation, suggesting that surviving SSCs have accumulated an increased load of DNA damage. Conclusions: SSCs revealed elevated levels of DSBs for weeks after radiation, and if these DSBs persist through differentiation to spermatozoa, this may have severe consequences for the genomic integrity of the fertilizing sperm.« less

  11. Colon Cancer Chemoprevention by Sage Tea Drinking: Decreased DNA Damage and Cell Proliferation.

    PubMed

    Pedro, Dalila F N; Ramos, Alice A; Lima, Cristovao F; Baltazar, Fatima; Pereira-Wilson, Cristina

    2016-02-01

    Salvia officinalis and some of its isolated compounds have been found to be preventive of DNA damage and increased proliferation in vitro in colon cells. In the present study, we used the azoxymethane model to test effects of S. officinalis on colon cancer prevention in vivo. The results showed that sage treatment reduced the number of ACF formed only if administered before azoxymethane injection, demonstrating that sage tea drinking has a chemopreventive effect on colorectal cancer. A decrease in the proliferation marker Ki67 and in H2 O2 -induced and azoxymethane-induced DNA damage to colonocytes and lymphocytes were found with sage treatment. This confirms in vivo the chemopreventive effects of S. officinalis. Taken together, our results show that sage treatment prevented initiation phases of colon carcinogenesis, an effect due, at least in part, to DNA protection, and reduced proliferation rates of colon epithelial cell that prevent mutations and their fixation through cell replication. These chemopreventive effects of S. officinalis on colon cancer add to the many health benefits attributed to sage and encourage its consumption. Copyright © 2015 John Wiley & Sons, Ltd.

  12. Lack of Duffy antigen expression is associated with organ damage in patients with sickle cell disease.

    PubMed

    Afenyi-Annan, Araba; Kail, Melanie; Combs, Martha R; Orringer, Eugene P; Ashley-Koch, Allison; Telen, Marilyn J

    2008-05-01

    The Duffy glycoprotein (Fy) on red blood cells (RBCs) has been hypothesized to promote clearance of inflammatory cytokines, which may play a role in the pathogenesis of vasoocclusion in sickle cell disease (SCD). Persons with the African-type Fy(a-b-) phenotype--whose RBCs lack expression of Duffy--may less efficiently clear inflammatory cytokines. Therefore, the Duffy-negative genotype may be associated with more severe disease among patients with SCD. Genotyping was performed on blood samples from 249 adult patients with HbSS at the Duffy gene (FY) locus GATA site (rs2814778) that determines RBC expression of Duffy antigens. Patients with discordant genotype and phenotype data were excluded (n = 12). Differences in demographic, clinical and laboratory findings, end-organ damage, and overall disease severity were compared between FY+ and FY- patients. Of the 237 patients studied, 174 (73%) were FY-. FY+ patients had a higher mean white blood cell (WBC) count (13.2 x 10(9) +/- 4.1 x 10(9)/L vs. 11.8 x 10(9) +/- 3.3 x 10(9)/L; p = 0.03) and higher rates of treatment with hydroxyurea (72% vs. 49%; p = 0.002). In contrast, FY- status was strongly associated with chronic organ damage (85% of FY- patients vs. 65% of FY+ patients; p = 0.018) and proteinuria (32% vs. 12%; p = 0.02). These associations remained, even after controlling for the effects of age and sex. Duffy genotype may be a potential biomarker for the development of end-organ damage in SCD, particularly kidney dysfunction. The association of both WBC counts and hydroxyurea use with Duffy expression provides another avenue for investigation of the biologic role of this protein.

  13. Platinum-induced kidney damage: Unraveling the DNA damage response (DDR) of renal tubular epithelial and glomerular endothelial cells following platinum injury.

    PubMed

    Krüger, Katharina; Thomale, Jürgen; Stojanović, Nikolina; Osmak, Maja; Henninger, Christian; Bormann, Stefanie; Fritz, Gerhard

    2015-03-01

    Platinum compounds are potent anticancer drugs but also evoke considerable normal tissue damage. Here, we elucidate the molecular mechanisms contributing to the nephrotoxic effects of cisplatin. We comparatively investigated the stress responses of rat kidney tubular (NRK-52E) and glomerular cells (RGE) following treatment with cisplatin (CisPt), oxaliplatin (OxaliPt) and carboplatin (CarboPt). To this end, cell viability, apoptosis, cell cycle progression, DNA damage response (DDR) and repair of DNA adducts were investigated. CisPt reduced the viability of tubular NRK-52E and glomerular RGE cells most efficiently. Cytotoxicity evoked by CarboPt occurred with a delay, which might be related to a retarded formation of Pt-(GpG) intrastrand crosslinks. RGE cells were more sensitive towards all platinum compounds than NRK-52E cells. Platinum drugs efficiently induced caspase-mediated apoptosis in tubular cells, while RGE cells favored G2/M arrest when treated with equitoxic platinum doses. Mitotic index of NKR-52E and RGE cells was worst affected by OxaliPt. Activation of the DDR was strikingly agent- and cell type-specific. Most comprehensive and substantial stimulation of DDR mechanisms was provoked by CisPt. Repair of Pt-(GpG) intrastrand crosslinks was best in RGE, which was reflected by high mRNA expression of nucleotide excision repair (NER) factors. There are substantial differences regarding the cause of sensitivity and mechanisms of DDR between tubular and glomerular cells following platinum injury. CisPt is the most potent stimulator of the DDR. We hypothesize that specific DNA adducts and thereby forcefully activated pro-toxic DDR mechanisms contribute to the exceptionally high acute nephrotoxicity of CisPt. Copyright © 2015 Elsevier B.V. All rights reserved.

  14. [Effect of astaxanthin on oxidative stress of red blood cells and peroxidation damage of membrane].

    PubMed

    Wang, Su-Ling; He, Lu-Jun; He, Tian-Bo; Han, Wei; Wang, Qie

    2015-04-01

    To explore the effect of astaxanthin (ASTA) on oxidative stress of intra- and extra- red blood cells during stored period and the protective function for cell membrane. The blood of volunteers was collected to prepare suspended red blood cells without leukocytes. Then the red blood cells were randomly divided into group A, group B, group C and group D. The ASTA was added into MAP preservation solution of group B, group C and group D, the final concentration of ASTA was 5, 10 and 20 µmol/L respectively. Group A was used as control group, in which only the dissolved liquid DMSO of ASTA was added. The red blood cells were stored in refrigerator at 2 °C-6 °C. On day 7, 14, 28 and day 42 of storage, the content of reactive oxygen species (ROS) in red blood cells was detected by fluorescence microplate reader. The content of malondialdehyde (MDA) was detected with TBA method. The content of hydrogen peroxide (H2O2) outside cell was detected with spectrophotometric method. The mean corpuscular volume(MCV) was detected with blood cell analyzer. The content of free hemoglobin(FHb) was detected with chemical colorimetry. The ROS, MDA, FHb and H2O2 levels in B, C and D groups were lower than those in control group during the stored period. On day 7 and 14 of storage, among group B, group C, group D and group A, the MCV showed no difference in comparison with control group. On day 28 and 42 of storage, the MCV in B, C and D groups was lower than that in control group. The ASTA can reduce the oxidative stress level of stored red blood cells inside and outside, relieve the peroxidation damage of cell membrane.

  15. Photooxidative damage in retinal pigment epithelial cells via GRP78 and the protective role of grape skin polyphenols.

    PubMed

    Zhao, Zhao; Sun, Tao; Jiang, Yun; Wu, Lijiang; Cai, Xiangzhong; Sun, Xiaodong; Sun, Xiangjun

    2014-12-01

    Blue light induced oxidative damage and ER stress are related to the pathogenesis of age-related macular degeneration (AMD). However, the mechanism of blue light-induced damage remained obscure. The objective of this work is to assess the photooxidative damage to retinal pigment epithelial cells (RPE) and oxidation-induced changes in expression of ER stress associated apoptotic proteins, and investigate the mechanism underlying the protective effects of grape skin extracts. To mimic lipofuscin-mediated photooxidation in vivo, ARPE-19 cells that accumulated A2E, one of lipofuscin fluorophores, were used as a model system to investigate the mechanism of photooxidative damage and the protective effects of grape skin polyphenols. Exposure of A2E containing ARPE-19 cells to blue light resulted in significant apoptosis and increases in levels of GRP78, CHOP, p-JNK, Bax, cleaved caspase-9, and cleaved caspase-3, indicating that photooxidative damage to RPE cells is mediated by the ER-stress-induced intrinsic apoptotic pathway. Cells in which GRP78 had been knocked down with shRNA were more vulnerable to photooxidative damage. Pre-treatment of blue-light-exposed A2E containing ARPE-19 cells, with grape skin extracts, inhibited apoptosis, in a dose dependent manner. Knockdown GRP78 blocked the protective effect of grape skin extracts.

  16. Relationship between Membrane Damage and Cell Death in Pressure-Treated Escherichia coli Cells: Differences between Exponential- and Stationary-Phase Cells and Variation among Strains

    PubMed Central

    Pagán, Rafael; Mackey, Bernard

    2000-01-01

    The relationship between membrane damage and loss of viability following pressure treatment was examined in Escherichia coli strains C9490, H1071, and NCTC 8003. These strains showed high, medium, and low resistance to pressure, respectively, in stationary phase but similar resistance to pressure in exponential phase. Loss of membrane integrity was measured as loss of osmotic responsiveness or as increased uptake of the fluorescent dye propidium iodide. In exponential-phase cells, loss of viability was correlated with a permanent loss of membrane integrity in all strains, whereas in stationary-phase cells, a more complicated picture emerged in which cell membranes became leaky during pressure treatment but resealed to a greater or lesser extent following decompression. Strain H1071 displayed a very unusual pressure response in stationary phase in which survival decreased to a minimum at 300 MPa but then increased at 400 to 500 MPa before decreasing again. Membranes were unable to reseal after treatment at 300 MPa but could do so after treatment at higher pressures. Membrane damage in this strain was thus typical of exponential-phase cells under low-pressure conditions but of stationary-phase cells under higher-pressure conditions. Heat shock treatment of strain H1071 cells increased pressure resistance under low-pressure conditions and also allowed membrane damage to reseal. Growth in the presence of IPTG (isopropyl-β-d-thiogalactopyranoside) increased resistance under high-pressure conditions. The mechanisms of inactivation may thus differ at high and low pressures. These studies support the view that membrane damage is an important event in the inactivation of bacteria by high pressure, but the nature of membrane damage and its relation to cell death may differ between species and phases of growth. PMID:10877775

  17. Apigenin attenuates streptozotocin-induced pancreatic β cell damage by its protective effects on cellular antioxidant defense.

    PubMed

    Wang, Ning; Yi, Wen Jing; Tan, Lu; Zhang, Jia Hui; Xu, Jiamin; Chen, Yi; Qin, Mengting; Yu, Shuang; Guan, Jing; Zhang, Rui

    2017-06-01

    Pancreatic beta cells are very sensitive to oxidative stress, which is one of the major causes of cell damages in diabetes. Growing interest has focused on the development of effective therapeutics to protect pancreatic cells from oxidative stress and searching for potentially protective antioxidants for treating diabetes. Apigenin, a plant-derived flavonoid, was investigated to determine whether it could protect rat insulinoma cell lines (RINm5F pancreatic beta cells) against streptozotocin (STZ)-induced oxidative damages and the mechanisms implicated. Our results showed that STZ treatment could induce oxidative stress and consequent cytotoxic effects in RINm5F cells. Pretreatment with apigenin effectively decreased the intracellular reactive oxygen species (ROS) production, attenuated cellular DNA damage, diminished lipid peroxidation, relieved protein carbonylation, and restored the cell apoptosis of pancreatic beta cells stressed by STZ. Our further experiments demonstrated that the beneficial effects of apigenin were related to ameliorate the loss of antioxidant enzymes of the STZ-treated cells in the level of gene transcription, protein expression, and enzyme activity. That suggested apigenin was not only a free radical scavenger but also a regulator to antioxidant defenses of pancreatic cells. Taken all together, our findings suggested that apigenin could attenuate the STZ-induced oxidative damages in pancreatic beta cells and might serve as a novel agent for the treatment of diabetes.

  18. 4β-Hydroxywithanolide E selectively induces oxidative DNA damage for selective killing of oral cancer cells.

    PubMed

    Tang, Jen-Yang; Huang, Hurng-Wern; Wang, Hui-Ru; Chan, Ya-Ching; Haung, Jo-Wen; Shu, Chih-Wen; Wu, Yang-Chang; Chang, Hsueh-Wei

    2018-03-01

    Reactive oxygen species (ROS) induction had been previously reported in 4β-hydroxywithanolide (4βHWE)-induced selective killing of oral cancer cells, but the mechanism involving ROS and the DNA damage effect remain unclear. This study explores the role of ROS and oxidative DNA damage of 4βHWE in the selective killing of oral cancer cells. Changes in cell viability, morphology, ROS, DNA double strand break (DSB) signaling (γH2AX foci in immunofluorescence and DSB signaling in western blotting), and oxidative DNA damage (8-oxo-2'deoxyguanosine [8-oxodG]) were detected in 4βHWE-treated oral cancer (Ca9-22) and/or normal (HGF-1) cells. 4βHWE decreased cell viability, changed cell morphology and induced ROS generation in oral cancer cells rather than oral normal cells, which were recovered by a free radical scavenger N-acetylcysteine (NAC). For immunofluorescence, 4βHWE also accumulated more of the DSB marker, γH2AX foci, in oral cancer cells than in oral normal cells. For western blotting, DSB signaling proteins such as γH2AX and MRN complex (MRE11, RAD50, and NBS1) were overexpressed in 4βHWE-treated oral cancer cells in different concentrations and treatment time. In the formamidopyrimidine-DNA glycolyase (Fpg)-based comet assay and 8-oxodG-based flow cytometry, the 8-oxodG expressions were higher in 4βHWE-treated oral cancer cells than in oral normal cells. All the 4βHWE-induced DSB and oxidative DNA damage to oral cancer cells were recovered by NAC pretreatment. Taken together, the 4βHWE selectively induced DSB and oxidative DNA damage for the ROS-mediated selective killing of oral cancer cells. © 2017 Wiley Periodicals, Inc.

  19. Effects of antidepressants on DSP4/CPT-induced DNA damage response in neuroblastoma SH-SY5Y cells

    PubMed Central

    Wang, Yan; Hilton, Benjamin A.; Cui, Kui; Zhu, Meng-Yang

    2015-01-01

    DNA damage is a form of cell stress and injury. Increased systemic DNA damage is related to the pathogenic development of neurodegenerative diseases. Depression occurs in a relatively high percentage of patients suffering from degenerative diseases, for whom antidepressants are often used to relieve depressive symptoms. However, few studies have attempted to elucidate why different groups of antidepressants have similar effects on relieving symptoms of depression. Previously, we demonstrated that neurotoxins N-(2-chloroethyl)-N-ethyl-2-bromobenzylamine (DSP4)- and camptothecin (CPT)-induced the DNA damage response in SH-SY5Y cells, and DSP4 caused cell cycle arrest which was predominately in the S-phase. The present study shows that CPT treatment also resulted in similar cell cycle arrest. Some classic antidepressants could reduce the DNA damage response induced by DSP4 or CPT in SH-SY5Y cells. Cell viability examination demonstrated that both DSP4 and CPT caused cell death, which was prevented by spontaneous administration of some tested antidepressants. Flow cytometric analysis demonstrated that a majority of the tested antidepressants protect cells from being arrested in S-phase. These results suggest that blocking the DNA damage response may be an important pharmacologic characteristic of antidepressants. Exploring the underlying mechanisms may allow for advances in the effort to improve therapeutic strategies for depression appearing in degenerative and psychiatric diseases. PMID:26038195

  20. Co-visualization of DNA damage and ion traversals in live mammalian cells using a fluorescent nuclear track detector

    PubMed Central

    Kodaira, Satoshi; Konishi, Teruaki; Kobayashi, Alisa; Maeda, Takeshi; Ahmad, Tengku Ahbrizal Farizal Tengku; Yang, Gen; Akselrod, Mark S.; Furusawa, Yoshiya; Uchihori, Yukio

    2015-01-01

    The geometric locations of ion traversals in mammalian cells constitute important information in the study of heavy ion-induced biological effect. Single ion traversal through a cellular nucleus produces complex and massive DNA damage at a nanometer level, leading to cell inactivation, mutations and transformation. We present a novel approach that uses a fluorescent nuclear track detector (FNTD) for the simultaneous detection of the geometrical images of ion traversals and DNA damage in single cells using confocal microscopy. HT1080 or HT1080–53BP1-GFP cells were cultured on the surface of a FNTD and exposed to 5.1-MeV/n neon ions. The positions of the ion traversals were obtained as fluorescent images of a FNTD. Localized DNA damage in cells was identified as fluorescent spots of γ-H2AX or 53BP1-GFP. These track images and images of damaged DNA were obtained in a short time using a confocal laser scanning microscope. The geometrical distribution of DNA damage indicated by fluorescent γ-H2AX spots in fixed cells or fluorescent 53BP1-GFP spots in living cells was found to correlate well with the distribution of the ion traversals. This method will be useful for evaluating the number of ion hits on individual cells, not only for micro-beam but also for random-beam experiments. PMID:25324538

  1. Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells

    PubMed Central

    Aytan, Nurgul; Karakas, Bahriye; Kurt, Asli Giray; Acikbas, Ufuk; Temel, Sehime Gulsun; Basaga, Huveyda

    2017-01-01

    Necrosis, apoptosis and autophagic cell death are the main cell death pathways in multicellular organisms, all with distinct and overlapping cellular and biochemical features. DNA damage may trigger different types of cell death in cancer cells but the molecular events governing the mode of cell death remain elusive. Here we showed that increased BH3-only protein BIK levels promoted cisplatin- and UV-induced mitochondrial apoptosis and biphasic ROS production in HCT-116 wild-type cells. Nonetheless, early single peak of ROS formation along with lysosomal membrane permeabilization and cathepsin activation regulated cisplatin- and UV-induced necrosis in p53-null HCT-116 cells. Of note, necrotic cell death in p53-null HCT-116 cells did not depend on BIK, mitochondrial outer membrane permeabilization or caspase activation. These data demonstrate how cancer cells with different p53 background respond to DNA-damaging agents by integrating distinct cell signaling pathways dictating the mode of cell death. PMID:28796811

  2. Biphasic ROS production, p53 and BIK dictate the mode of cell death in response to DNA damage in colon cancer cells.

    PubMed

    Kutuk, Ozgur; Aytan, Nurgul; Karakas, Bahriye; Kurt, Asli Giray; Acikbas, Ufuk; Temel, Sehime Gulsun; Basaga, Huveyda

    2017-01-01

    Necrosis, apoptosis and autophagic cell death are the main cell death pathways in multicellular organisms, all with distinct and overlapping cellular and biochemical features. DNA damage may trigger different types of cell death in cancer cells but the molecular events governing the mode of cell death remain elusive. Here we showed that increased BH3-only protein BIK levels promoted cisplatin- and UV-induced mitochondrial apoptosis and biphasic ROS production in HCT-116 wild-type cells. Nonetheless, early single peak of ROS formation along with lysosomal membrane permeabilization and cathepsin activation regulated cisplatin- and UV-induced necrosis in p53-null HCT-116 cells. Of note, necrotic cell death in p53-null HCT-116 cells did not depend on BIK, mitochondrial outer membrane permeabilization or caspase activation. These data demonstrate how cancer cells with different p53 background respond to DNA-damaging agents by integrating distinct cell signaling pathways dictating the mode of cell death.

  3. Chlorella protects against hydrogen peroxide-induced pancreatic β-cell damage.

    PubMed

    Lin, Chia-Yu; Huang, Pei-Jane; Chao, Che-Yi

    2014-12-01

    Oxidative stress has been implicated in the etiology of pancreatic β-cell dysfunction and diabetes. Studies have shown that chlorella could be important in health promotion or disease prevention through its antioxidant capacity. However, whether chlorella has a cytoprotective effect in pancreatic β-cells remains to be elucidated. We investigated the protective effects of chlorella on H2O2-induced oxidative damage in INS-1 (832/13) cells. Chlorella partially restored cell viability after H2O2 toxicity. To further investigate the effects of chlorella on mitochondria function and cellular oxidative stress, we analyzed mitochondria membrane potential, ATP concentrations, and cellular levels of reactive oxygen species (ROS). Chlorella prevented mitochondria disruption and maintained cellular ATP levels after H2O2 toxicity. It also normalized intracellular levels of ROS to that of control in the presence of H2O2. Chlorella protected cells from apoptosis as indicated by less p-Histone and caspase 3 activation. In addition, chlorella not only enhanced glucose-stimulated insulin secretion (GSIS), but also partially restored the reduced GSIS after H2O2 toxicity. Our results suggest that chlorella is effective in amelioration of cellular oxidative stress and destruction, and therefore protects INS-1 (832/13) cells from H2O2-induced apoptosis and increases insulin secretion. Chlorella should be studied for use in the prevention or treatment of diabetes.

  4. Regulatory networks integrating cell cycle control with DNA damage checkpoints and double-strand break repair.

    PubMed

    Langerak, Petra; Russell, Paul

    2011-12-27

    Double-strand breaks (DSBs), arising from exposure to exogenous clastogens or as a by-product of endogenous cellular metabolism, pose grave threats to genome integrity. DSBs can sever whole chromosomes, leading to chromosomal instability, a hallmark of cancer. Healing broken DNA takes time, and it is therefore essential to temporarily halt cell division while DSB repair is underway. The seminal discovery of cyclin-dependent kinases as master regulators of the cell cycle unleashed a series of studies aimed at defining how the DNA damage response network delays cell division. These efforts culminated with the identification of Cdc25, the protein phosphatase that activates Cdc2/Cdk1, as a critical target of the checkpoint kinase Chk1. However, regulation works both ways, as recent studies have revealed that Cdc2 activity and cell cycle position determine whether DSBs are repaired by non-homologous end-joining or homologous recombination (HR). Central to this regulation are the proteins that initiate the processing of DNA ends for HR repair, Mre11-Rad50-Nbs1 protein complex and Ctp1/Sae2/CtIP, and the checkpoint kinases Tel1/ATM and Rad3/ATR. Here, we review recent findings and provide insight on how proteins that regulate cell cycle progression affect DSB repair, and, conversely how proteins that repair DSBs affect cell cycle progression.

  5. DNA damage response signaling in lung adenocarcinoma A549 cells following gamma and carbon beam irradiation.

    PubMed

    Ghosh, Somnath; Narang, Himanshi; Sarma, Asitikantha; Krishna, Malini

    2011-11-01

    Carbon beams (5.16MeV/u, LET=290keV/μm) are high linear energy transfer (LET) radiation characterized by higher relative biological effectiveness than low LET radiation. The aim of the current study was to determine the signaling differences between γ-rays and carbon ion-irradiation. A549 cells were irradiated with 1Gy carbon or γ-rays. Carbon beam was found to be three times more cytotoxic than γ-rays despite the fact that the numbers of γ-H2AX foci were same. Percentage of cells showing ATM/ATR foci were more with γ-rays however number of foci per cell were more in case of carbon irradiation. Large BRCA1 foci were found in all carbon irradiated cells unlike γ-rays irradiated cells and prosurvival ERK pathway was activated after γ-rays irradiation but not carbon. The noteworthy finding of this study is the early phase apoptosis induction by carbon ions. In the present study in A549 lung adenocarcinoma, authors conclude that despite activation of same repair molecules such as ATM and BRCA1, differences in low and high LET damage responses might be due to their distinct macromolecular complexes rather than their individual activation and the activation of cytoplasmic pathways such as ERK, whether it applies to all the cell lines need to be further explored. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Effective blue light photodynamic therapy does not affect cutaneous langerhans cell number or oxidatively damage DNA.

    PubMed

    Ramaswamy, Preethi; Powers, Jennifer G; Bhawan, Jag; Polyak, Ildiko; Gilchrest, Barbara A

    2014-09-01

    Photodynamic therapy (PDT) using aminolevulinic acid (ALA) with blue light or red light is effective for treating actinic keratoses (AKs). However, immunosuppression follows red light PDT, raising the spectre of skin cancer promotion in treated skin. To determine whether broad-area short incubation (BASI)-ALA-PDT using blue light immunosuppression immunosuppresses treated skin. Patients were evaluated clinically and by standardized facial biopsies of non-AK skin before, 24 hours and 1 month after customary blue light BASI-ALA-PDT. All biopsies were stained for markers of epidermal atypia and Langerhans cells (LCs); and at 24 hours to detect oxidative DNA damage. Patients had an 81% reduction in AKs and slight improvement in clinical and histologic signs of photoaging after 1 month. The biopsied chronically photodamaged skin without clinically detectable AKs showed no effect of PDT on the LC number, distribution, or morphology; and no oxidative DNA damage, in contrast to the changes reported after customary red light PDT. Customary blue light BASI-ALA-PDT does not affect the LC number or produce oxidative DNA damage, the sequelae of red light PDT responsible for immunosuppression in treated skin.

  7. Comparative Study of Corneal Endothelial Cell Damage after Femtosecond Laser Assisted Deep Stromal Dissection

    PubMed Central

    Liu, Ting; Zhang, Jingjing; Sun, Dapeng; Sui, Wenjie; Zhang, Yangyang; Li, Dongfang; Chen, Zhaoli

    2014-01-01

    Purpose. To find a relatively safe designed stromal bed thickness to avoid endothelial damage for lamellar keratoplasty with an Allegretto Wavelight FS200 femtosecond laser. Methods. Twelve rabbits were randomly divided into 50 μm and 150 μm groups according to the anticipated residue stromal bed thickness preparation with a femtosecond laser. Six rabbits without laser cutting were used as a control group. Central endothelial images were analyzed with in vivo confocal microscopy and scanning electron microscopy. The apoptosis of endothelium was evaluated with Hoechst 33342 staining and a TUNEL assay. Results. The endothelium of the 50 μm group had extensive injuries upon in vivo confocal and scanning electron microscopic observation, and minor injuries were observed in the 150 μm group. Moreover, more apoptotic cells were observed in the 50 μm group. Conclusions. When using a FS200 femtosecond laser assisted anterior lamellar keratoplasty, there was minor endothelium damage with a 150 μm stromal bed, and a more than 150 μm thickness stromal bed design may prevent the damage of corneal endothelium. PMID:25114918

  8. Single-cell resolution mapping of neuronal damage in acute focal cerebral ischemia using thallium autometallography

    PubMed Central

    Stöber, Franziska; Baldauf, Kathrin; Ziabreva, Iryna; Harhausen, Denise; Zille, Marietta; Neubert, Jenni; Reymann, Klaus G; Scheich, Henning; Dirnagl, Ulrich; Schröder, Ulrich H; Wunder, Andreas; Goldschmidt, Jürgen

    2014-01-01

    Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K+-probe thallium (Tl+) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl+ after crossing the blood–brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl+ uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl+ uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl+ uptake. At 24 hours, the areas of reduced Tl+uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of 201TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K+ metabolism and prediction of tissue viability in cerebral ischemia. PMID:24129748

  9. Single-cell resolution mapping of neuronal damage in acute focal cerebral ischemia using thallium autometallography.

    PubMed

    Stöber, Franziska; Baldauf, Kathrin; Ziabreva, Iryna; Harhausen, Denise; Zille, Marietta; Neubert, Jenni; Reymann, Klaus G; Scheich, Henning; Dirnagl, Ulrich; Schröder, Ulrich H; Wunder, Andreas; Goldschmidt, Jürgen

    2014-01-01

    Neuronal damage shortly after onset or after brief episodes of cerebral ischemia has remained difficult to assess with clinical and preclinical imaging techniques as well as with microscopical methods. We here show, in rodent models of middle cerebral artery occlusion (MCAO), that neuronal damage in acute focal cerebral ischemia can be mapped with single-cell resolution using thallium autometallography (TlAMG), a histochemical technique for the detection of the K(+)-probe thallium (Tl(+)) in the brain. We intravenously injected rats and mice with thallium diethyldithiocarbamate (TlDDC), a lipophilic chelate complex that releases Tl(+) after crossing the blood-brain barrier. We found, within the territories of the affected arteries, areas of markedly reduced neuronal Tl(+) uptake in all animals at all time points studied ranging from 15 minutes to 24 hours after MCAO. In large lesions at early time points, areas with neuronal and astrocytic Tl(+) uptake below thresholds of detection were surrounded by putative penumbral zones with preserved but diminished Tl(+) uptake. At 24 hours, the areas of reduced Tl(+)uptake matched with areas delineated by established markers of neuronal damage. The results suggest the use of (201)TlDDC for preclinical and clinical single-photon emission computed tomography (SPECT) imaging of hyperacute alterations in brain K(+) metabolism and prediction of tissue viability in cerebral ischemia.

  10. Neuroglobin Is an Endogenous Neuroprotectant for Retinal Ganglion Cells against Glaucomatous Damage

    PubMed Central

    Wei, Xin; Yu, Zhanyang; Cho, Kin-Sang; Chen, Huihui; Malik, Muhammad Taimur A.; Chen, Xiaoming; Lo, Eng H.; Wang, Xiaoying; Chen, Dong F.

    2011-01-01

    Neuroglobin (NGB), a newly discovered member of the globin superfamily, may regulate neuronal survival under hypoxia or oxidative stress. Although NGB is greatly expressed in retinal neurons, the biological functions of NGB in retinal diseases remain largely unknown. We investigated the role of NGB in an experimental model of glaucoma, a neurodegenerative disorder that usually involves elevation of intraocular pressure (IOP). Elevated IOP is thought to induce oxidative stress in retinal ganglion cells (RGCs), thereby causing RGC death and, eventually, blindness. We found that NGB plays a critical role in increasing RGC resistance to ocular hypertension and glaucomatous damage. Elevation of IOP stimulated a transient up-regulation of endogenous NGB in RGCs. Constitutive overexpression of NGB in transgenic mice prevented RGC damage induced by glutamate cytotoxicity in vitro and/or by chronic IOP elevation in vivo. Moreover, overexpression of NGB attenuated ocular hypertension-induced superoxide production and the associated decrease in ATP levels in mice, suggesting that NGB acts as an endogenous neuroprotectant to reduce oxidative stress and improve mitochondrial function, thereby promoting RGC survival. Thus, NGB may modulate RGC susceptibility to glaucomatous neural damage. Manipulating the expression and bioactivity of NGB may represent a novel therapeutic strategy for glaucoma. PMID:21967817

  11. Ca2+-Permeable AMPARs Mediate Glutamatergic Transmission and Excitotoxic Damage at the Hair Cell Ribbon Synapse.

    PubMed

    Sebe, Joy Y; Cho, Soyoun; Sheets, Lavinia; Rutherford, Mark A; von Gersdorff, Henrique; Raible, David W

    2017-06-21

    We report functional and structural evidence for GluA2-lacking Ca 2+ -permeable AMPARs (CP-AMPARs) at the mature hair cell ribbon synapse. By using the methodological advantages of three species (of either sex), we demonstrate that CP-AMPARs are present at the hair cell synapse in an evolutionarily conserved manner. Via a combination of in vivo electrophysiological and Ca 2+ imaging approaches in the larval zebrafish, we show that hair cell stimulation leads to robust Ca 2+ influx into afferent terminals. Prolonged application of AMPA caused loss of afferent terminal responsiveness, whereas blocking CP-AMPARs protects terminals from excitotoxic swelling. Immunohistochemical analysis of AMPAR subunits in mature rat cochlea show regions within synapses lacking the GluA2 subunit. Paired recordings from adult bullfrog auditory synapses demonstrate that CP-AMPARs mediate a major component of glutamatergic transmission. Together, our results support the importance of CP-AMPARs in mediating transmission at the hair cell ribbon synapse. Further, excess Ca 2+ entry via CP-AMPARs may underlie afferent terminal damage following excitotoxic challenge, suggesting that limiting Ca 2+ levels in the afferent terminal may protect against cochlear synaptopathy associated with hearing loss. SIGNIFICANCE STATEMENT A single incidence of noise overexposure causes damage at the hair cell synapse that later leads to neurodegeneration and exacerbates age-related hearing loss. A first step toward understanding cochlear neurodegeneration is to identify the cause of initial excitotoxic damage to the postsynaptic neuron. Using a combination of immunohistochemical, electrophysiological, and Ca 2+ imaging approaches in evolutionarily divergent species, we demonstrate that Ca 2+ -permeable AMPARs (CP-AMPARs) mediate glutamatergic transmission at the adult auditory hair cell synapse. Overexcitation of the terminal causes Ca 2+ accumulation and swelling that can be prevented by blocking CP

  12. Dihydromyricetin protects endothelial cells from hydrogen peroxide-induced oxidative stress damage by regulating mitochondrial pathways.

    PubMed

    Hou, Xiaolong; Tong, Qing; Wang, Wenqing; Xiong, Wei; Shi, Chunyang; Fang, Jianguo

    2015-06-01

    Dihydromyricetin (DMY) is the most abundant ingredient in vine tea. Here, we investigated the cytoprotective effects and possible mechanisms of DMY on hydrogen peroxide (H2O2)-induced oxidative stress damage in human umbilical vein endothelial cells (HUVECs). The percentage of cell viability was evaluated using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. We determined the antioxidant properties of DMY by measuring the activity of superoxide dismutase (SOD) and malondialdehyde (MDA). Flow cytometry was used to measure apoptosis in HUVECs that were double stained with Hoechst 33342 and propidium iodide (PI). The generation of intracellular reactive oxygen species (ROS) was measured in 2',7'-dichlorofluorescin diacetate (DCFH-DA)-loaded HUVECs using a fluorescent microscope. Moreover, the expression of apoptosis-related proteins was determined by Western blotting. In addition, the release of nitric oxide (NO) was analyzed using a commercial kit. HUVECs treated with H2O2 had a notable decrease in cell viability that was attenuated when cells were pretreated with DMY (37.5-300μM). DMY pretreatment significantly attenuated H2O2-induced apoptosis in HUVECs and inhibited intracellular ROS overproduction. Finally, pretreatment of cells with DMY prior to H2O2 exposure resulted in the inhibition of p53 activation, followed by the regulation of the expression of Bcl-2 and Bax, the release of cytochrome c, the cleavage (activation) of caspase-9 and caspase-3, and then the suppression of PARP cleavage in H2O2-induced HUVECs. Our study is the first to report that DMY can protect HUVECs from oxidative stress damage, an effect that is mediated by the mitochondrial apoptotic pathways. Copyright © 2015 Elsevier Inc. All rights reserved.

  13. YAP and TAZ in epithelial stem cells: A sensor for cell polarity, mechanical forces and tissue damage.

    PubMed

    Elbediwy, Ahmed; Vincent-Mistiaen, Zoé I; Thompson, Barry J

    2016-07-01

    The YAP/TAZ family of transcriptional co-activators drives cell proliferation in epithelial tissues and cancers. Yet, how YAP and TAZ are physiologically regulated remains unclear. Here we review recent reports that YAP and TAZ act primarily as sensors of epithelial cell polarity, being inhibited when cells differentiate an apical membrane domain, and being activated when cells contact the extracellular matrix via their basal membrane domain. Apical signalling occurs via the canonical Crumbs/CRB-Hippo/MST-Warts/LATS kinase cascade to phosphorylate and inhibit YAP/TAZ. Basal signalling occurs via Integrins and Src family kinases to phosphorylate and activate YAP/TAZ. Thus, YAP/TAZ is localised to the nucleus in basal stem/progenitor cells and cytoplasm in differentiated squamous cells or columnar cells. In addition, other signals such as mechanical forces, tissue damage and possibly receptor tyrosine kinases (RTKs) can influence MST-LATS or Src family kinase activity to modulate YAP/TAZ activity. © 2016 The Authors BioEssays Published by WILEY Periodicals, Inc.

  14. Resveratrol Induces Premature Senescence in Lung Cancer Cells via ROS-Mediated DNA Damage

    PubMed Central

    Luo, Hongmei; Yang, Aimin; Schulte, Bradley A.; Wargovich, Michael J.; Wang, Gavin Y.

    2013-01-01

    Resveratrol (RV) is a natural component of red wine and grapes that has been shown to be a potential chemopreventive and anticancer agent. However, the molecular mechanisms underlying RV's anticancer and chemopreventive effects are incompletely understood. Here we show that RV treatment inhibits the clonogenic growth of non-small cell lung cancer (NSCLC) cells in a dose-dependent manner. Interestingly, the tumor-suppressive effect of low dose RV was not associated with any significant changes in the expression of cleaved PARP and activated caspase-3, suggesting that low dose RV treatment may suppress tumor cell growth via an apoptosis-independent mechanism. Subsequent studies reveal that low dose RV treatment induces a significant increase in senescence-associated β–galactosidase (SA-β-gal) staining and elevated expression of p53 and p21 in NSCLC cells. Furthermore, we show that RV-induced suppression of lung cancer cell growth is associated with a decrease in the expression of EF1A. These results suggest that RV may exert its anticancer and chemopreventive effects through the induction of premature senescence. Mechanistically, RV-induced premature senescence correlates with increased DNA double strand breaks (DSBs) and reactive oxygen species (ROS) production in lung cancer cells. Inhibition of ROS production by N-acetylcysteine (NAC) attenuates RV-induced DNA DSBs and premature senescence. Furthermore, we show that RV treatment markedly induces NAPDH oxidase-5 (Nox5) expression in both A549 and H460 cells, suggesting that RV may increase ROS generation in lung cancer cells through upregulating Nox5 expression. Together, these findings demonstrate that low dose RV treatment inhibits lung cancer cell growth via a previously unappreciated mechanism, namely the induction of premature senescence through ROS-mediated DNA damage. PMID:23533664

  15. Protective effects of edaravone against cisplatin-induced hair cell damage in zebrafish.

    PubMed

    Hong, Seok Jin; Im, Gi Jung; Chang, Jiwon; Chae, Sung Won; Lee, Seung Hoon; Kwon, Soon Young; Jung, Hak Hyun; Chung, Ah Young; Park, Hae Chul; Choi, June

    2013-06-01

    Edaravone is known to have a potent free radical scavenging effect. The objective of the present study was to evaluate the effects of edaravone on cisplatin-induced ototoxicity in transgenic zebrafish (Brn3C: EGFP). Five day post-fertilization zebrafish larvae were exposed to 1000 μM cisplatin and 50 μM, 100 μM, 250 μM, 500 μM, 750 μM, and 1000 μM concentrations of edaravone for 4h. Hair cells within neuromasts of the supraorbital (SO1 and SO2), otic (O1), and occipital (OC1) lateral lines were analyzed by fluorescence microscopy and confocal microscopy (n=10). Hair cell survival was calculated as a percentage of the hair cells in the control group that were not exposed to cisplatin. Ultrastructural changes were evaluated using scanning electron microscopy and transmission electron microscopy. Edaravone protected cisplatin-induced hair cell loss of neuromasts (edaravone 750 μM: 8.7 ± 1.5 cells, cisplatin 1000 μM only: 3.7 ± 0.9 cells; n=10, p<0.0001) and decreased the Terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end labeling (TUNEL) reaction. Structures of mitochondria and hair cell within neuromasts in ultrastructural analysis were preserved in zebrafish exposed to 1000 μM cisplatin and 750 μM edaravone for 4h. Edaravone attenuated cisplatin-induced hair cell damage in zebrafish. The results of the current study suggest that cisplatin induces apoptosis, and the apoptotic cell death can be prevented by treatment with edaravone in zebrafish. Copyright © 2013 Elsevier Ireland Ltd. All rights reserved.

  16. Response of thyroid follicular cells to gamma irradiation compared to proton irradiation. I. Initial characterization of DNA damage, micronucleus formation, apoptosis, cell survival, and cell cycle phase redistribution

    NASA Technical Reports Server (NTRS)

    Green, L. M.; Murray, D. K.; Bant, A. M.; Kazarians, G.; Moyers, M. F.; Nelson, G. A.; Tran, D. T.

    2001-01-01

    The RBE of protons has been assumed to be equivalent to that of photons. The objective of this study was to determine whether radiation-induced DNA and chromosome damage, apoptosis, cell killing and cell cycling in organized epithelial cells was influenced by radiation quality. Thyroid-stimulating hormone-dependent Fischer rat thyroid cells, established as follicles, were exposed to gamma rays or proton beams delivered acutely over a range of physical doses. Gamma-irradiated cells were able to repair DNA damage relatively rapidly so that by 1 h postirradiation they had approximately 20% fewer exposed 3' ends than their counterparts that had been irradiated with proton beams. The persistence of free ends of DNA in the samples irradiated with the proton beam implies that either more initial breaks or a quantitatively different type of damage had occurred. These results were further supported by an increased frequency of chromosomal damage as measured by the presence of micronuclei. Proton-beam irradiation induced micronuclei at a rate of 2.4% per gray, which at 12 Gy translated to 40% more micronuclei than in comparable gamma-irradiated cultures. The higher rate of micronucleus formation and the presence of larger micronuclei in proton-irradiated cells was further evidence that a qualitatively more severe class of damage had been induced than was induced by gamma rays. Differences in the type of damage produced were detected in the apoptosis assay, wherein a significant lag in the induction of apoptosis occurred after gamma irradiation that did not occur with protons. The more immediate expression of apoptotic cells in the cultures irradiated with the proton beam suggests that the damage inflicted was more severe. Alternatively, the cell cycle checkpoint mechanisms required for recovery from such damage might not have been invoked. Differences based on radiation quality were also evident in the alpha components of cell survival curves (0.05 Gy(-1) for gamma rays, 0

  17. RADIATION DAMAGE IN MUSCLE CELL MEMBRANES AND REGULATION OF CELL METABOLISM

    SciTech Connect

    Portela, A.; Perez, J.C.; Stewart, P.

    1963-10-31

    The absence of detectable early effects on frog striated muscle of low doses (under 10,000 r) of x rays has been reported. Effects of higher (100,000 r) doses include prolonged relaxation time, more rapid fatigue, and decreased ATP and glycogen content, but increased potassium and sodium effluxes, sodium influx, and oxygen consumption, compared with non-irradiated controls. Twitch latency and rise time are not changed. Magnesium-activated ATPase activity of homogenates or mitochondrial suspensions decreases immediately after irradiation, but increases with time more rapidly than in controls. The biochemical findings suggest uncoupling of oxidative phosphorylation, while the ion flux data suggest increasedmore » membrane permeability. An hypothesis is suggested that irradiation results in membrane damage, both in the sarcoplasma membrane, thereby increasing ion permeabilities, and in the mitochondrial structure, thereby interfering with oxidative phosphorylation and reducing ATP production. A decrease in membrane potential during irradiation to about 90% of its control value has already been reported. The observed increases in Na and K fluxes after irradiation explain these findings. At these radiation levels, our data do not require any hynothesis of early direct damage to the contractile mechanism per se. (auth)« less

  18. Solid Oxide Fuel Cells damage mechanisms due to Ni-YSZ re-oxidation: Case of the Anode Supported Cell

    NASA Astrophysics Data System (ADS)

    Laurencin, J.; Delette, G.; Morel, B.; Lefebvre-Joud, F.; Dupeux, M.

    The effects of Ni-YSZ cermet re-oxidation in anode supported Solid Oxide Fuel Cells (SOFCs) have been investigated. Damage mechanisms have been studied in both cases of direct oxidation in air (i.e., fuel shutdown) or by an ionic current (i.e., fuel starvation). Direct oxidation tests show that the electrolyte cracks for a conversion degree of Ni into NiO ranging between ∼58 and ∼71%. This failure mode has been modelled considering both the bulk expansion of the cermet induced by the transformation of the Ni phase and the change of mechanical stresses in the multilayered cell. In the case of fuel starvation, a thin layer of the cermet was electrochemically re-oxidised at 800 °C and then reduced under a hydrogen stream. This 'redox' cycle was repeated until the degradation of the cell. The evolution of the impedance diagrams recorded after each cycle suggests that the cermet damages in an area close to anode/electrolyte interface. The mechanical modelling states that a delamination can occur along the interface between the Anode Functional Layer (AFL) and the Anode Current Collector (ACC) substrate. This theoretical result confirms the experimental trends observed by impedance spectroscopy.

  19. miR-638 suppresses DNA damage repair by targeting SMC1A expression in terminally differentiated cells

    PubMed Central

    He, Mingyang; Lin, Yi; Tang, Yunlan; Liu, Yi; Zhou, Weiwei; Li, Chuang; Sun, Guihong; Guo, Mingxiong

    2016-01-01

    The reduction of DNA damage repair capacity in terminally differentiated cells may be involved in sensitivity to cancer chemotherapy drugs; however, the underlying molecular mechanism is still not fully understood. Herein, we evaluated the role of miR-638 in the regulation of DNA damage repair in terminally differentiated cells. Our results show that miR-638 expression was up-regulated during cellular terminal differentiation and involved in mediating DNA damage repair processes. Results from a luciferase reporting experiment show that structural maintenance of chromosomes (SMC)1A was a potential target of miR-638; this was verified by western blot assays during cell differentiation and DNA damage induction. Overexpression of miR-638 enhanced the sensitivity of cancer cells to cisplatin, thus reducing cell viability in response to chemotherapy drug treatment. Furthermore, miR-638 overexpression affected DNA damage repair processes by interfering with the recruitment of the DNA damage repair-related protein, γH2AX, to DNA break sites. These findings indicate that miR-638 might act as a sensitizer in cancer chemotherapy and accompany chemotherapy drugs to enhance chemotherapeutic efficacy and to improve the chance of recovery from cancer. PMID:27405111

  20. Pim-3 contributes to radioresistance through regulation of the cell cycle and DNA damage repair in pancreatic cancer cells

    SciTech Connect

    Chen, Xiang-Yuan; Wang, Zhen; Li, Bei

    2016-04-22

    Resistance of cancer cells to chemoradiotherapy is a major clinical problem in pancreatic cancer treatment. Therefore, understanding the molecular basis of cellular resistance and identifying novel targets are essential for improving treatment efficacy for pancreatic cancer patients. Previous studies have demonstrated a significant role for Pim-3 in pancreatic cancer survival against gemcitabine-induced genotoxic stress. Here, we observed that radiation treatment enhanced Pim-3 expression in human pancreatic cancer cells in vitro. Stable overexpression of Pim-3 in pancreatic cancer cells significantly protected cells against radiation treatment by attenuating G2/M phase cell cycle arrest and DNA damage response. Silencing of Pim-3 expression significantly elevatedmore » the phosphorylation of histone variant H2AX, a marker of DNA double strand breaks, and decreased the activation of ataxia-telangiectasia-mutated (ATM) kinase, along with its downstream targets, eventually enhancing the radiosensitivity of human pancreatic cancer cells in vitro and in vivo. Hence, we demonstrated a novel function for Pim-3 in human pancreatic cancer cell survival against radiation. Targeting Pim-3 may be a promising way to improve treatment efficacy in combination with radiotherapy in human pancreatic cancer. - Highlights: • This is first study to demonstrate that Pim-3 is endogenously induced by ionizing radiation in pancreatic cancer cells, and Pim-3 overexpression enhanced radioresistance of pancreatic cancer cells both in vitro and in vivo. • This is first study to provide evidence that radioresistance induced by Pim-3 is mainly attributed to Pim-3 induces activation of ATM, which subsequently activates checkpoint 1, leading to amplification of DNA repair through cell cycle arrest and DNA repair pathways. • This is first study to indicate that targeting Pim-3 may be a promising strategy to provide better treatment efficacy in combination with radiotherapy in human

  1. Mesenchymal stem cells sense mitochondria released from damaged cells as danger signals to activate their rescue properties

    PubMed Central

    Mahrouf-Yorgov, Meriem; Augeul, Lionel; Da Silva, Claire Crola; Jourdan, Maud; Rigolet, Muriel; Manin, Sylvie; Ferrera, René; Ovize, Michel; Henry, Adeline; Guguin, Aurélie; Meningaud, Jean-Paul; Dubois-Randé, Jean-Luc; Motterlini, Roberto; Foresti, Roberta; Rodriguez, Anne-Marie

    2017-01-01

    Mesenchymal stem cells (MSCs) protect tissues against cell death induced by ischemia/reperfusion insults. This therapeutic effect seems to be controlled by physiological cues released by the local microenvironment following injury. Recent lines of evidence indicate that MSC can communicate with their microenvironment through bidirectional exchanges of mitochondria. In particular, in vitro and in vivo studies report that MSCs rescue injured cells through delivery of their own mitochondria. However, the role of mitochondria conveyed from somatic cells to MSC remains unknown. By using a co-culture system consisting of MSC and distressed somatic cells such as cardiomyocytes or endothelial cells, we showed that mitochondria from suffering cells acted as danger-signaling organelles that triggered the anti-apoptotic function of MSC. We demonstrated that foreign somatic-derived mitochondria were engulfed and degraded by MSC, leading to induction of the cytoprotective enzyme heme oxygenase-1 (HO-1) and stimulation of mitochondrial biogenesis. As a result, the capacity of MSC to donate their mitochondria to injured cells to combat oxidative stress injury was enhanced. We found that similar mechanisms – activation of autophagy, HO-1 and mitochondrial biogenesis – occurred after exposure of MSC to exogenous mitochondria isolated from somatic cells, strengthening the idea that somatic mitochondria alert MSC of a danger situation and subsequently promote an adaptive reparative response. In addition, the cascade of events triggered by the transfer of somatic mitochondria into MSC was recapitulated in a model of myocardial infarction in vivo. Specifically, MSC engrafted into infarcted hearts of mice reduced damage, upregulated HO-1 and increased mitochondrial biogenesis, while inhibition of mitophagy or HO-1 failed to protect against cardiac apoptosis. In conclusion, our study reveals a new facet about the role of mitochondria released from dying cells as a key environmental

  2. The cell cycle phases of DNA damage and repair initiated by topoisomerase II-targeting chemotherapeutic drugs.

    PubMed

    Potter, Alan J; Rabinovitch, Peter S

    2005-05-02

    Although cytostasis and cytotoxicity induced by cancer chemotherapy drugs targeting topoisomerase II (topoII) arise in specific cell cycle phases, it is unknown whether the drug-initiated DNA damage triggering these responses, or the repair (reversal) of this damage, differs between cell cycle phases or between drug classes. Accordingly, we used a flow cytometric alkaline unwinding assay to measure DNA damage (strand breakage (SB)) and SB repair in each cell cycle compartment of human cancer cell lines treated with clinically relevant concentrations of doxorubicin, daunomycin, etoposide, and mitoxantrone. We found that treated HeLa and A549 cells exhibited the greatest SB in G2/M phase, the least in G1 phase, and generally an intermediate amount in S phase. The cell cycle phase specificity of the DNA damage appeared to be predictive of the cell cycle phase of growth arrest. Furthermore, it appeared to be dependent on topoIIalpha expression as the extent of SB did not differ between cell cycle compartments in topoIIalpha-diminished A549(VP)28 cells. HeLa cells were apparently unable to repair doxorubicin-initiated SB. The rate of repair of etoposide-initiated SB in HeLa cells and of mitoxantrone-initiated SB in HeLa and A549 cells was similar in each cell cycle compartment. In A549 cells, the rate of repair of doxorubicin and etoposide-initiated SB differed between cell cycle phases. Overall, these results indicate that the cell cycle phase specificity of cytostasis and cytotoxicity induced in tumor cells by topoII-targeting drugs may be directly related to the cell cycle phase specificity of the drug-initiated DNA damage. Analysis by cell cycle compartment appears to clarify some of the intercellular heterogeneity in the extent of drug-initiated DNA damage and cytotoxicity previously observed in cancer cells analyzed as a single population; this approach might be useful in resolving inconsistent results reported in investigations of tumor cell topoII content versus

  3. Displacement Damage Effects in Solar Cells: Mining Damage From the Microelectronics and Photonics Test Bed Space Experiment

    NASA Technical Reports Server (NTRS)

    Hardage, Donna (Technical Monitor); Walters, R. J.; Morton, T. L.; Messenger, S. R.

    2004-01-01

    The objective is to develop an improved space solar cell radiation response analysis capability and to produce a computer modeling tool which implements the analysis. This was accomplished through analysis of solar cell flight data taken on the Microelectronics and Photonics Test Bed experiment. This effort specifically addresses issues related to rapid technological change in the area of solar cells for space applications in order to enhance system performance, decrease risk, and reduce cost for future missions.

  4. Human amnion tissue injected with human umbilical cord mesenchymal stem cells repairs damaged sciatic nerves in rats★

    PubMed Central

    Li, Dehua; Wang, Changhui; Shan, Wei; Zeng, Ruixia; Fang, Yan; Wang, Pan

    2012-01-01

    Human umbilical cord mesenchymal stem cells, incorporated into an amnion carrier tubes, were assessed for nerve regeneration potential in a rat nerve defect model. Damaged nerves were exposed to human amnion carriers containing either human umbilical cord mesenchymal stem cell (cell transplantation group) or saline (control group). At 8, 12, 16 and 20 weeks after cell implantation, the sciatic functional index was higher in the cell transplantation group compared with the control group. Furthermore, electrophysiological examination showed that threshold stimulus and maximum stimulus intensity gradually decreased while compound action potential amplitude gradually increased. Hematoxylin-eosin staining showed that regenerating nerve fibers were arranged in nerve tracts in the cell transplantation group and connective tissue between nerve tracts and amnion tissue reduced over time. Gastrocnemius muscle cell diameter, wet weight and restoration ratio were increased. These data indicate that transplanted human umbilical cord mesenchymal stem cells, using the amnion tube connection method, promote restoration of damaged sciatic nerves in rats. PMID:25624800

  5. A comparative study of chemically induced DNA damage in isolated human and rat testicular cells.

    PubMed

    Bjørge, C; Brunborg, G; Wiger, R; Holme, J A; Scholz, T; Dybing, E; Søderlund, E J

    1996-01-01

    Testicular cells prepared from human organ transplant donors or from Wistar rats were used to compare 15 known reproductive toxicants with respect to their ability to induce DNA damage, measured as single-strand DNA breaks and alkali labile sites (ssDNA breaks) with alkaline filter elution. The compounds tested included various categories of chemicals (i.e., pesticides, industrial chemicals, cytostatics, and mycotoxins) most of which are directly acting genotoxicants (i.e., reacting with DNA either spontaneously or via metabolic activation). In addition, a few indirect genotoxic and nongenotoxic reproductive toxicants were included. Six of the chemicals induced no significant levels of ssDNA breaks in human and rat testicular cells; methoxychlor (10 to 100 microM, human and rat), benomyl (10 to 100 microM, human and rat), thiotepa (10 to 1000 microM, human and rat), cisplatin (30 to 1000 microM, human; 100 to 1000 microM, rat), Cd2+ (30 to 1000 microM, human; 100 to 1000 microM, rat), and acrylonitrile (30 to 1000 microM, human; 30 to 300 microM, rat). Four chemicals induced significant levels of ssDNA breaks in testicular cells from both species: styrene oxide (> or = 100 microM, rat and human), 1,2-dibromoethane (EDB) (> or = 100 microM, rat; 1000 microM human), thiram (> or = 30 microM, rat; > or = 100 microM, human), and chlordecone (300 microM, rat; > or = 300 microM, human). Finally, five chemicals induced ssDNA breaks in one of the two species. Four chemicals induced significant ssDNA breaks in rat testicular cells only: 1,2-dibromo-3-chloropropane (DBCP) (> or = 10 microM), 1,3-dinitrobenzene (1,3-DNB) (> or = 300 microM), Cr6+ (1000 microM), and aflatoxin B1 (> or = 100 microM), the last two of these produced only a minor positive response. One chemical, acrylamide, induced a marginal increase in ssDNA breaks in human at 1000 microM, but not in rat testicular cells. Although based on a limited number of donors, the data indicate a close correlation between

  6. DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer.

    PubMed

    Sears, Catherine R; Cooney, Sean A; Chin-Sinex, Helen; Mendonca, Marc S; Turchi, John J

    2016-04-01

    Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24h after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism. Published by Elsevier B.V.

  7. DNA damage response (DDR) pathway engagement in cisplatin radiosensitization of non-small cell lung cancer

    PubMed Central

    Sears, Catherine R.; Cooney, Sean A.; Chin-Sinex, Helen; Mendonca, Marc S.; Turchi, John J.

    2016-01-01

    Non-small cell lung cancers (NSCLC) are commonly treated with a platinum-based chemotherapy such as cisplatin (CDDP) in combination with ionizing radiation (IR). Although clinical trials have demonstrated that the combination of CDDP and IR appear to be synergistic in terms of therapeutic efficacy, the mechanism of synergism remains largely uncharacterized. We investigated the role of the DNA damage response (DDR) in CDDP radiosensitization using two NSCLC cell lines. Using clonogenic survival assays, we determined that the cooperative cytotoxicity of CDDP and IR treatment is sequence dependent, requiring administration of CDDP prior to IR (CDDP-IR). We identified and interrogated the unique time and agent-dependent activation of the DDR in NSCLC cells treated with cisplatin-IR combination therapy. Compared to treatment with CDDP or IR alone, CDDP-IR combination treatment led to persistence of γH2Ax foci, a marker of DNA double-strand breaks (DSB), for up to 24 hours after treatment. Interestingly, pharmacologic inhibition of DDR sensor kinases revealed the persistence of γ-H2Ax foci in CDDP-IR treated cells is independent of kinase activation. Taken together, our data suggest that delayed repair of DSBs in NSCLC cells treated with CDDP-IR contributes to CDDP radiosensitization and that alterations of the DDR pathways by inhibition of specific DDR kinases can augment CDDP-IR cytotoxicity by a complementary mechanism. PMID:26991853

  8. Obatoclax potentiates the cytotoxic effect of cytarabine on acute myeloid leukemia cells by enhancing DNA damage.

    PubMed

    Xie, Chengzhi; Edwards, Holly; Caldwell, J Timothy; Wang, Guan; Taub, Jeffrey W; Ge, Yubin

    2015-02-01

    Resistance to cytarabine and anthracycline-based chemotherapy is a major cause of treatment failure for acute myeloid leukemia (AML) patients. Overexpression of Bcl-2, Bcl-xL, and/or Mcl-1 has been associated with chemoresistance in AML cell lines and with poor clinical outcome of AML patients. Thus, inhibitors of anti-apoptotic Bcl-2 family proteins could be novel therapeutic agents. In this study, we investigated how clinically achievable concentrations of obatoclax, a pan-Bcl-2 inhibitor, potentiate the antileukemic activity of cytarabine in AML cells. MTT assays in AML cell lines and diagnostic blasts, as well as flow cytometry analyses in AML cell lines revealed synergistic antileukemic activity between cytarabine and obatoclax. Bax activation was detected in the combined, but not the individual, drug treatments. This was accompanied by significantly increased loss of mitochondrial membrane potential. Most importantly, in AML cells treated with the combination, enhanced early induction of DNA double-strand breaks (DSBs) preceded a decrease of Mcl-1 levels, nuclear translocation of Bcl-2, Bcl-xL, and Mcl-1, and apoptosis. These results indicate that obatoclax enhances cytarabine-induced apoptosis by enhancing DNA DSBs. This novel mechanism provides compelling evidence for the clinical use of BH3 mimetics in combination with DNA-damaging agents in AML and possibly a broader range of malignancies. Copyright © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.

  9. In vitro PAMAM, phosphorus and viologen-phosphorus dendrimers prevent rotenone-induced cell damage.

    PubMed

    Milowska, Katarzyna; Szwed, Aleksandra; Zablocka, Maria; Caminade, Anne-Marie; Majoral, Jean-Pierre; Mignani, Serge; Gabryelak, Teresa; Bryszewska, Maria

    2014-10-20

    We have investigated whether polyamidoamine (PAMAM), phosphorus (pd) and viologen-phosphorus (vpd) dendrimers can prevent damage to embryonic mouse hippocampal cells (mHippoE-18) caused by rotenone, which is used as a pesticide, insecticide, and as a nonselective piscicide, that works by interfering with the electron transport chain in mitochondria. Several basic aspects, such as cell viability, production of reactive oxygen species and changes in mitochondrial transmembrane potential, were analyzed. mHippoE-18 cells were treated with these structurally different dendrimers at 0.1μM. A 1h incubation with dendrimers was followed by the addition of rotenone at 1μM, and a further 24h incubation. PAMAM, phosphorus and viologen-phosphorus dendrimers all increased cell viability (reduced cell death-data need to be compared with untreated controls). A lower level of reactive oxygen species and a favorable effect on mitochondrial system were found with PAMAM and viologen-phosphorus dendrimers. These results indicate reduced toxicity in the presence of dendrimers. Copyright © 2014 Elsevier B.V. All rights reserved.

  10. Kinetics of the Cell Biological Changes Occurring in the Progression of DNA Damage-Induced Senescence

    PubMed Central

    Cho, Sohee; Park, Jihoon; Hwang, Eun Seong

    2011-01-01

    Cellular senescence is characterized by cell-cycle arrest accompanied by various cell biological changes. Although these changes have been heavily relied on as senescence markers in numerous studies on senescence and its intervention, their underlying mechanisms and relationship to each other are poorly understood. Furthermore, the depth and the reversibility of those changes have not been addressed previously. Using flow cytometry coupled with confocal microscopy and Western blotting, we quantified various senescence-associated cellular changes and determined their time course profiles in MCF-7 cells undergoing DNA damage-induced senescence. The examined properties changed with several different kinetics patterns. Autofluorescence, side scattering, and the mitochondria content increased progressively and linearly. Cell volume, lysosome content, and reactive oxygen species (ROS) level increased abruptly at an early stage. Meanwhile, senescence associated β-galactosidase activity increased after a lag of a few days. In addition, during the senescence progression, lysosomes exhibited a loss of integrity, which may have been associated with the accumulation of ROS. The finding that various senescence phenotypes matured at different rates with different lag times suggests multiple independent mechanisms controlling the expression of senescence phenotypes. This type of kinetics study would promote the understanding of how cells become fully senescent and facilitate the screening of methods that intervene in cellular senescence. PMID:21533552

  11. S-methylmethionine reduces cell membrane damage in higher plants exposed to low-temperature stress.

    PubMed

    Rácz, Ilona; Páldi, Emil; Szalai, Gabriella; Janda, Tibor; Pál, Magdolna; Lásztity, Demeter

    2008-09-29

    S-methylmethionine (SMM), an important intermediate compound in the sulphur metabolism, can be found in various quantities in majority of plants. The experiments were designed to determine the extent to which SMM is able to preserve cell membrane integrity or reduce the degree of membrane damage in the course of low-temperature stress. By measuring electrolyte leakage (EL), it was proved that SMM treatment reduced cell membrane damage, and thus EL, during low-temperature stress in both the leaves and roots of peas, maize, soy beans and eight winter wheat varieties with different levels of frost resistance. Investigations on the interaction between SMM and polyamine biosynthesis revealed that SMM increased the quantities of agmatine (Agm) and putrescine (Put) as well as that of spermidine (Spd), while it had no effect on the quantity of spermine (Spn). Using a specific inhibitor, methylglyoxal-bis-guanyl hydrazone (MGBG), it was proved that the polyamine metabolic pathway starting from methionine played no role in the synthesis of Spd or Spn, so there must be an alternative pathway for the synthesis of SMM-induced polyamines.

  12. Cell and gene therapy for arrhythmias: Repair of cardiac conduction damage

    PubMed Central

    Xiao, Yong-Fu

    2011-01-01

    Action potentials generated in the sinoatrial node (SAN) dominate the rhythm and rate of a healthy human heart. Subsequently, these action potentials propagate to the whole heart via its conduction system. Abnormalities of impulse generation and/or propagation in a heart can cause arrhythmias. For example, SAN dysfunction or conduction block of the atrioventricular node can lead to serious bradycardia which is currently treated with an implanted electronic pacemaker. On the other hand, conduction damage may cause reentrant tachyarrhythmias which are primarily treated pharmacologically or by medical device-based therapies, including defibrillation and tissue ablation. However, drug therapies sometimes may not be effective or are associated with serious side effects. Device-based therapies for cardiac arrhythmias, even with well developed technology, still face inadequacies, limitations, hardware complications, and other challenges. Therefore, scientists are actively seeking other alternatives for antiarrhythmic therapy. In particular, cells and genes used for repairing cardiac conduction damage/defect have been investigated in various studies both in vitro and in vivo. Despite the complexities of the excitation and conduction systems of the heart, cell and gene-based strategies provide novel alternatives for treatment or cure of cardiac arrhythmias. This review summarizes some highlights of recent research progress in this field. PMID:22783301

  13. Radiosensitization of mammalian cells by misonidazole and oxygen: DNA damage exposed by Micrococcus luteus enzymes

    SciTech Connect

    Skov, K.A.; Palcic, B.; Skarsgard, L.D.

    1979-09-01

    When misonidazole is present during irradiation of hypoxic mammalian cells, an enhancement of single-strand breaks (SSB) in DNA is observed. Oxygen also enhances SSB, presumably in a manner similar to that of misonidazole. The dose-modifying factor (DMF) for 15 mM misonidazole was found to be 3.4, compared to an oxygen enhancement ratio (OER) of 3.5. Another class of DNA damage, namely, sites exposed by an extract of Micrococcus luteus, was examined. Radiation-induced M. luteus extract-sensitive sites (MLS) were also found to be enhanced by the presence of misonidazole or molecular oxygen. The DMF for this damage by 15 mM misonidazolemore » was 1.6 while the OER was 2.5. The ratio of MLS to SSB is approximately 1.25 under hypoxia, 0.9 in the presence of oxygen, and 0.6 in the presence of 15 mM misonidazole under hypoxic conditions. Incubation with misonidazole under conditions which are toxic to mammalian cells (37/sup 0/C, hypoxia), and which result in many SSB, produces no detectable lesions sensitive to the M. luteus extract.« less

  14. Interstrand Cross-Links Induce DNA Synthesis in Damaged and Undamaged Plasmids in Mammalian Cell Extracts

    PubMed Central

    Li, Lei; Peterson, Carolyn A.; Lu, Xiaoyan; Wei, Ping; Legerski, Randy J.

    1999-01-01

    Mammalian cell extracts have been shown to carry out damage-specific DNA repair synthesis induced by a variety of lesions, including those created by UV and cisplatin. Here, we show that a single psoralen interstrand cross-link induces DNA synthesis in both the damaged plasmid and a second homologous unmodified plasmid coincubated in the extract. The presence of the second plasmid strongly stimulates repair synthesis in the cross-linked plasmid. Heterologous DNAs also stimulate repair synthesis to variable extents. Psoralen monoadducts and double-strand breaks do not induce repair synthesis in the unmodified plasmid, indicating that such incorporation is specific to interstrand cross-links. This induced repair synthesis is consistent with previous evidence indicating a recombinational mode of repair for interstrand cross-links. DNA synthesis is compromised in extracts from mutants (deficient in ERCC1, XPF, XRCC2, and XRCC3) which are all sensitive to DNA cross-linking agents but is normal in extracts from mutants (XP-A, XP-C, and XP-G) which are much less sensitive. Extracts from Fanconi anemia cells exhibit an intermediate to wild-type level of activity dependent upon the complementation group. The DNA synthesis deficit in ERCC1- and XPF-deficient extracts is restored by addition of purified ERCC1-XPF heterodimer. This system provides a biochemical assay for investigating mechanisms of interstrand cross-link repair and should also facilitate the identification and functional characterization of cellular proteins involved in repair of these lesions. PMID:10409751

  15. Equivalent electron fluence for solar proton damage in GaAs shallow junction cells

    NASA Technical Reports Server (NTRS)

    Wilson, J. W.; Stock, L. V.

    1984-01-01

    The short-circuit current reduction in GaAs shallow junction heteroface solar cells was calculated according to a simplified solar cell damage model in which the nonuniformity of the damage as a function of penetration depth is treated explicitly. Although the equivalent electron fluence was not uniquely defined for low-energy monoenergetic proton exposure, an equivalent electron fluence is found for proton spectra characteristic of the space environment. The equivalent electron fluence ratio was calculated for a typical large solar flare event for which the proton spectrum is PHI(sub p)(E) = A/E(p/sq. cm) where E is in MeV. The equivalent fluence ratio is a function of the cover glass shield thickness or the corresponding cutoff energy E(sub c). In terms of the cutoff energy, the equivalent 1 MeV electron fluence ratio is r(sub p)(E sub c) = 10(9)/E(sub c)(1.8) where E(sub c) is in units of KeV.

  16. Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells

    PubMed Central

    Modi, Prexy; Balakrishnan, Kumudha; Yang, Qingshan; Wierda, William G.; Keating, Michael J.; Gandhi, Varsha

    2017-01-01

    Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL. PMID:28187444

  17. Idelalisib and bendamustine combination is synergistic and increases DNA damage response in chronic lymphocytic leukemia cells.

    PubMed

    Modi, Prexy; Balakrishnan, Kumudha; Yang, Qingshan; Wierda, William G; Keating, Michael J; Gandhi, Varsha

    2017-03-07

    Idelalisib is a targeted agent that potently inhibits PI3Kδ which is exclusively expressed in hematological cells. Bendamustine is a well-tolerated cytotoxic alkylating agent which has been extensively used for treatment of chronic lymphocytic leukemia (CLL). Both these agents are FDA-approved for CLL. To increase the potency of idelalisib and bendamustine, we tested their combination in primary CLL lymphocytes. While each compound alone produced a moderate response, combination at several concentrations resulted in synergistic cytotoxicity. Idelalisib enhanced the bendamustine-mediated DNA damage/repair response, indicated by the phosphorylation of ATM, Chk2, and p53. Each drug alone activated γH2AX but combination treatment further increased the expression of this DNA damage marker. Compared with the control, idelalisib treatment decreased global RNA synthesis, resulting in a decline of early-response and short-lived MCL1 transcripts. In concert, there was a decline in total Mcl-1 protein in CLL lymphocytes. Isogenic mouse embryonic fibroblasts lacking MCL1 had higher sensitivity to bendamustine alone or in combination compared to MCL1 proficient cells. Collectively, these data indicate that bendamustine and idelalisib combination therapy should be investigated for treating patients with CLL.

  18. Circulating endothelial cells demonstrate an attenuation of endothelial damage by minimizing the extracorporeal circulation.

    PubMed

    Skrabal, Christian A; Choi, Yeong H; Kaminski, Alexander; Steiner, Michael; Kundt, Guenther; Steinhoff, Gustav; Liebold, Andreas

    2006-08-01

    Detachment of endothelial cells may represent serious injury of the endothelium after cardiopulmonary bypass. We investigated whether the extent of endothelial injury is related to the type of cardiopulmonary bypass system used (conventional or minimized) and determined circulating endothelial cells as well as von Willebrand factor and soluble thrombomodulin. Twenty patients scheduled for elective coronary bypass grafting were randomly assigned to either the minimal extracorporeal circulation system or the standard cardiopulmonary bypass. Ten healthy volunteers served as controls. Circulating endothelial cells per milliliter of full blood were perioperatively determined by immunomagnetic cell separation technique. Endothelial plasma markers were measured by enzyme-linked immunosorbent assay. Preoperative circulating endothelial cell numbers did not differ between the experimental groups, but were significantly higher than in the healthy controls (18.6 +/- 5.6 vs 7.2 +/- 3.8, P < .001). At 6 hours, circulating endothelial cell numbers increased significantly compared with baseline in both experimental groups and peaked at 12 hours after cardiopulmonary bypass initiation, each time with significantly lower values in the minimal extracorporeal circulation group (6 hours: 44.0 +/- 9.9 vs 29.6 +/- 9.8, P = .007; 12 hours: 48.1 +/- 6.8 vs 31.8 +/- 7.1, P < .001). Likewise, von Willebrand factor and soluble thrombomodulin postoperatively increased in both groups with a tendency toward lower levels in the minimal extracorporeal circulation group. Although circulating endothelial cells gradually declined, continually with lower numbers in the minimal extracorporeal circulation group, the endothelial plasma markers remained elevated during observation time. Circulating endothelial cel