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• W2004828905 abstract "Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 μm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53. Oxidative stress and persistent DNA damage response contribute to cellular senescence, a degeneration process critically involving ataxia telangiectasia-mutated (ATM) and p53. Selenoprotein H (SelH), a nuclear selenoprotein, is proposed to carry redox and transactivation domains. To determine the role of SelH in genome maintenance, shRNA knockdown was employed in human normal and immortalized cell lines. SelH shRNA MRC-5 diploid fibroblasts under ambient O2 displayed a distinct profile of senescence including β-galactosidase expression, autofluorescence, growth inhibition, and ATM pathway activation. Such senescence phenotypes were alleviated in the presence of ATM kinase inhibitors, by p53 shRNA knockdown, or by maintaining the cells under 3% O2. During the course of 5-day recovery, the induction of phospho-ATM on Ser-1981 and γH2AX by H2O2 treatment (20 μm) subsided in scrambled shRNA but exacerbated in SelH shRNA MRC-5 cells. Results from clonogenic assays demonstrated hypersensitivity of SelH shRNA HeLa cells to paraquat and H2O2, but not to hydroxyurea, neocarzinostatin, or camptothecin. While SelH mRNA expression was induced by H2O2 treatment, SelH-GFP did not mobilize to sites of oxidative DNA damage. The glutathione level was lower in SelH shRNA than scrambled shRNA HeLa cells, and the H2O2-induced cell death was rescued in the presence of N-acetylcysteine, a glutathione precursor. Altogether, SelH protects against cellular senescence to oxidative stress through a genome maintenance pathway involving ATM and p53. Cellular senescence restricts cell proliferation through permanent withdrawal from the cell cycle and plays dual physiological roles (1Rodier F. Campisi J. Four faces of cellular senescence.J. Cell Biol. 2011; 192: 547-556Crossref PubMed Scopus (1393) Google Scholar, 2Fumagalli M. Rossiello F. Clerici M. Barozzi S. Cittaro D. Kaplunov J.M. Bucci G. Dobreva M. Matti V. Beausejour C.M. Herbig U. Longhese M.P. d'Adda di Fagagna F. Telomeric DNA damage is irreparable and causes persistent DNA-damage-response activation.Nat. Cell Biol. 2012; 14: 355-365Crossref PubMed Scopus (526) Google Scholar, 3Mallette F.A. Ferbeyre G. The DNA damage signaling pathway connects oncogenic stress to cellular senescence.Cell Cycle. 2007; 6: 1831-1836Crossref PubMed Scopus (107) Google Scholar, 4Takahashi A. Ohtani N. Yamakoshi K. Iida S. Tahara H. Nakayama K. Nakayama K.I. Ide T. Saya H. Hara E. Mitogenic signalling and the p16INK4a-Rb pathway cooperate to enforce irreversible cellular senescence.Nat. Cell Biol. 2006; 8: 1291-1297Crossref PubMed Scopus (389) Google Scholar). While cellular senescence can curb tumorigenesis at the precancerous stage and control fibrosis during cutaneous healing early in life (5Halazonetis T.D. Gorgoulis V.G. Bartek J. An oncogene-induced DNA damage model for cancer development.Science. 2008; 319: 1352-1355Crossref PubMed Scopus (1397) Google Scholar, 6Jun J.I. Lau L.F. The matricellular protein CCN1 induces fibroblast senescence and restricts fibrosis in cutaneous wound healing.Nat. Cell Biol. 2010; 12: 676-685Crossref PubMed Scopus (614) Google Scholar), it contributes to cellular and tissue aging and age-related disorders later in life (7Ressler S. Bartkova J. Niederegger H. Bartek J. Scharffetter-Kochanek K. Jansen-Dürr P. Wlaschek M. p16INK4A is a robust in vivo biomarker of cellular aging in human skin.Aging Cell. 2006; 5: 379-389Crossref PubMed Scopus (381) Google Scholar, 8Voghel G. Thorin-Trescases N. Farhat N. Nguyen A. Villeneuve L. Mamarbachi A.M. Fortier A. Perrault L.P. Carrier M. Thorin E. Cellular senescence in endothelial cells from atherosclerotic patients is accelerated by oxidative stress associated with cardiovascular risk factors.Mech Ageing Dev. 2007; 128: 662-671Crossref PubMed Scopus (118) Google Scholar, 9Wang C. Jurk D. Maddick M. Nelson G. Martin-Ruiz C. von Zglinicki T. DNA damage response and cellular senescence in tissues of aging mice.Aging Cell. 2009; 8: 311-323Crossref PubMed Scopus (467) Google Scholar, 10Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9363-9367Crossref PubMed Scopus (5707) Google Scholar). Although replicative senescence is induced by gradual telomere dysfunction in proliferating cells, stress-induced senescence occurs in essentially any cell type. Reactive oxygen species (ROS) 3The abbreviations used are: ROSreactive oxygen speciesPCNAproliferating cell nuclear antigenSelHselenoprotein HATMataxia telangiectasia-mutatedNACN-acetylcysteineCM-H2DCFDA5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetateSA-β-galsenescence-associated β-galactosidasemBCLmonochlorobimane. can induce the formation of oxidative and broken DNA, resulting in persistent activation of the DNA damage response and senescence if left unrepaired. Ataxia telangiectasia-mutated (ATM) is a key DNA damage response kinase coordinating checkpoint and senescence responses. ATM is activated by either DNA breaks or oxidative stress (11Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (1073) Google Scholar, 12Guo Z. Kozlov S. Lavin M.F. Person M.D. Paull T.T. ATM activation by oxidative stress.Science. 2010; 330: 517-521Crossref PubMed Scopus (797) Google Scholar), and plays an essential role in the senescence response by phosphorylating and stabilizing p53 (13Canman C.E. Lim D.S. Cimprich K.A. Taya Y. Tamai K. Sakaguchi K. Appella E. Kastan M.B. Siliciano J.D. Activation of the ATM kinase by ionizing radiation and phosphorylation of p53.Science. 1998; 281: 1677-1679Crossref PubMed Scopus (1703) Google Scholar, 14Bartkova J. Rezaei N. Liontos M. Karakaidos P. Kletsas D. Issaeva N. Vassiliou L.V. Kolettas E. Niforou K. Zoumpourlis V.C. Takaoka M. Nakagawa H. Tort F. Fugger K. Johansson F. Sehested M. Andersen C.L. Dyrskjot L. Ørntoft T. Lukas J. Kittas C. Helleday T. Halazonetis T.D. Bartek J. Gorgoulis V.G. Oncogene-induced senescence is part of the tumorigenesis barrier imposed by DNA damage checkpoints.Nature. 2006; 444: 633-637Crossref PubMed Scopus (1497) Google Scholar, 15Suzuki M. Suzuki K. Kodama S. Yamashita S. Watanabe M. Persistent amplification of DNA damage signal involved in replicative senescence of normal human diploid fibroblasts.Oxid. Med. Cell Longev. 2012; 2012: 310534Crossref PubMed Scopus (14) Google Scholar). reactive oxygen species proliferating cell nuclear antigen selenoprotein H ataxia telangiectasia-mutated N-acetylcysteine 5-(and-6)-chloromethyl-2′, 7′-dichlorodihydrofluorescein diacetate senescence-associated β-galactosidase monochlorobimane. In mammals, most selenoproteins carry antioxidative functions (16Kryukov G.V. Castellano S. Novoselov S.V. Lobanov A.V. Zehtab O. Guigó R. Gladyshev V.N. Characterization of mammalian selenoproteomes.Science. 2003; 300: 1439-1443Crossref PubMed Scopus (1839) Google Scholar, 17Lu J. Holmgren A. Selenoproteins.J. Biol. Chem. 2009; 284: 723-727Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). Selenoprotein expression requires the essential trace element, selenium. Selenoprotein H (SelH), glutathione peroxidase-1, selenoprotein W, and selenoprotein M are more sensitive than other selenoproteins to body selenium fluctuations (18Sunde R.A. Molecular biomarker panels for assessment of selenium status in rats.Exp. Biol. Med. 2010; 235: 1046-1052Crossref PubMed Scopus (27) Google Scholar, 19Raines A.M. Sunde R.A. Selenium toxicity but not deficient or super-nutritional selenium status vastly alters the transcriptome in rodents.BMC Genomics. 2011; 12: 26Crossref PubMed Scopus (71) Google Scholar, 20Kipp A.P. Banning A. van Schothorst E.M. Méplan C. Coort S.L. Evelo C.T. Keijer J. Hesketh J. Brigelius-Flohé R. Marginal selenium deficiency down-regulates inflammation-related genes in splenic leukocytes of the mouse.J. Nutr. Biochem. 2012; 23: 1170-1177Crossref PubMed Scopus (28) Google Scholar). Tissue-specific knockout of selenocysteine tRNA for global suppression of selenoproteins in epidermal cells or osteo-chondroprogenitor cells renders the mice susceptible to age-related disorders including alopecia and bone abnormality (21Sengupta A. Lichti U.F. Carlson B.A. Ryscavage A.O. Gladyshev V.N. Yuspa S.H. Hatfield D.L. Selenoproteins are essential for proper keratinocyte function and skin development.PLoS ONE. 2010; 5: e12249Crossref PubMed Scopus (71) Google Scholar, 22Downey C.M. Horton C.R. Carlson B.A. Parsons T.E. Hatfield D.L. Hallgrímsson B. Jirik F.R. Osteo-chondroprogenitor-specific deletion of the selenocysteine tRNA gene, Trsp, leads to chondronecrosis and abnormal skeletal development: a putative model for Kashin-Beck disease.PLoS Genet. 2009; 5: e1000616Crossref PubMed Scopus (89) Google Scholar). These observations are consistent with an estimation linking eleven selenoproteins to aging or age-related disorders (23McCann J.C. Ames B.N. Adaptive dysfunction of selenoproteins from the perspective of the triage theory: why modest selenium deficiency may increase risk of diseases of aging.FASEB J. 2011; 25: 1793-1814Crossref PubMed Scopus (87) Google Scholar). SelH is a thioredoxin-like nuclear protein exhibiting glutathione peroxidase activity (24Novoselov S.V. Kryukov G.V. Xu X.M. Carlson B.A. Hatfield D.L. Gladyshev V.N. Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern.J. Biol. Chem. 2007; 282: 11960-11968Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar). Furthermore, the homologue of human SelH in Drosophila is critical for embryogenesis through its antioxidative activity (25Morozova N. Forry E.P. Shahid E. Zavacki A.M. Harney J.W. Kraytsberg Y. Berry M.J. Antioxidant function of a novel selenoprotein in Drosophila melanogaster.Genes Cells. 2003; 8: 963-971Crossref PubMed Scopus (41) Google Scholar). Studies of human SelH in HT22 mouse neuronal cells have implicated this selenoprotein in the protection against UVB-induced apoptosis and as a transactivator for GSH biosynthesis (26Ben Jilani K.E. Panee J. He Q. Berry M.J. Li P.A. Overexpression of selenoprotein H reduces Ht22 neuronal cell death after UVB irradiation by preventing superoxide formation.Int. J. Biol. Sci. 2007; 3: 198-204Crossref PubMed Google Scholar, 27Panee J. Stoytcheva Z.R. Liu W. Berry M.J. Selenoprotein H is a redox-sensing high mobility group family DNA-binding protein that up-regulates genes involved in glutathione synthesis and phase II detoxification.J. Biol. Chem. 2007; 282: 23759-23765Abstract Full Text Full Text PDF PubMed Scopus (89) Google Scholar, 28Mendelev N. Witherspoon S. Li P.A. Overexpression of human selenoprotein H in neuronal cells ameliorates ultraviolet irradiation-induced damage by modulating cell signaling pathways.Exp. Neurol. 2009; 220: 328-334Crossref PubMed Scopus (26) Google Scholar, 29Mendelev N. Mehta S.L. Witherspoon S. He Q. Sexton J.Z. Li P.A. Upregulation of human selenoprotein H in murine hippocampal neuronal cells promotes mitochondrial biogenesis and functional performance.Mitochondrion. 2011; 11: 76-82Crossref PubMed Scopus (38) Google Scholar). Nonetheless, a role of SelH in the senescence response to DNA damage and oxidative stress has not been explored. Because SelH expression is enriched in nucleoli, and this organelle has been proposed as a stress-sensing center in the nucleus (24Novoselov S.V. Kryukov G.V. Xu X.M. Carlson B.A. Hatfield D.L. Gladyshev V.N. Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern.J. Biol. Chem. 2007; 282: 11960-11968Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar, 30Lewinska A. Wnuk M. Grzelak A. Bartosz G. Nucleolus as an oxidative stress sensor in the yeast Saccharomyces cerevisiae.Redox Rep. 2010; 15: 87-96Crossref PubMed Scopus (19) Google Scholar, 31Mayer C. Bierhoff H. Grummt I. The nucleolus as a stress sensor: JNK2 inactivates the transcription factor TIF-IA and down-regulates rRNA synthesis.Genes Dev. 2005; 19: 933-941Crossref PubMed Scopus (176) Google Scholar), we hypothesized that SelH protects against oxidative stress through genome maintenance and the limitation of cellular senescence. Thus, we stably knocked down SelH expression in human normal diploid fibroblasts and cancerous cells to evaluate their cellular and biochemical responses to various DNA-damaging agents. Our results suggested a new role of SelH specifically in the cellular response to oxidative stress that suppresses senescence and gatekeeps genomic integrity in a manner depending on ATM and p53. The MRC-5 diploid lung fibroblasts (Coriell Institute, Camden, NJ), HeLa cervical cancer cells (ATCC, Manassas, VA), and HCT116 colorectal adenocarcinoma cells complemented with hMLH1 (HCT116+hMLH1) (32Koi M. Umar A. Chauhan D.P. Cherian S.P. Carethers J.M. Kunkel T.A. Boland C.R. Human chromosome 3 corrects mismatch repair deficiency and microsatellite instability and reduces N-methyl-N′-nitro-N-nitrosoguanidine tolerance in colon tumor cells with homozygous hMLH1 mutation.Cancer Res. 1994; 54: 4308-4312PubMed Google Scholar, 33Yanamadala S. Ljungman M. Potential role of MLH1 in the induction of p53 and apoptosis by blocking transcription on damaged DNA templates.Mol. Cancer Res. 2003; 1: 747-754PubMed Google Scholar) were cultured as described previously in 20% or 3% O2 incubators (34Wu M. Kang M.M. Schoene N.W. Cheng W.H. Selenium compounds activate early barriers of tumorigenesis.J. Biol. Chem. 2010; 285: 12055-12062Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Qi Y. Schoene N.W. Lartey F.M. Cheng W.H. Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.J. Biol. Chem. 2010; 285: 33010-33017Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). However, no additional selenium was supplemented in the current study. Because selenium inevitably appears in FBS, a typical cell culture medium containing 10–15% FBS can support selenoprotein expression at nutritional level. By analysis, the batch of FBS used in this study contains selenium at 355 nm. N-Acetylcysteine (NAC), a GSH precursor, was dissolved in water. KU 60019 and KU 55933 (Tocris, Ellisville, MO) were dissolved in DMSO. All chemicals were from Sigma-Aldrich unless otherwise indicated. SelH and SelH2 shRNA sequences targeting the 3′ SelH mRNA at nucleotides 333–353 and 503–523 were designed based on Invitrogen Block-itTM RNAi designer, and the human non-target scrambled sequence (5′-CCTAAGGTTAAGTCGCCCTCGC-3′) was adapted from Addgene Organization. The lentiviral particles containing shRNA cassette were produced by BLOCK-iT™ Lentiviral RNAi Expression System (Life Technologies) and used to infect cells in culture. After viral infection (1 day), recovery (1 day), and blasticidin selection (14 days), 12 viable clones were picked from each viral infection and sub-cultured to confirm the knockdown efficiency by using quantitative RT-PCR. Human TaqMan probes (FAM-tagged) and primers were purchased from Applied Biosystems using inventoried TaqMan gene expression assays: SelH (Hs00415057_m1) and GAPDH (Hs99999905_m1). Cells containing SelH or SelH2 shRNA sequences expressed ∼80% less SelH mRNA as compared with scrambled shRNA cells. A viable colony is defined as one containing more than 50 cells. Passage 2 SelH shRNA MRC-5 cells were employed for experiments unless otherwise indicated. p53 shRNA knockdown was performed as described previously (36Wu M. Wu R.T. Wang T.T. Cheng W.H. Role for p53 in Selenium-Induced Senescence.J. Agric. Food Chem. 2011; 59: 11882-11887Crossref PubMed Scopus (13) Google Scholar). Levels of intracellular ROS were assessed by using 5-(and-6)-chloromethyl-2′,7′-dichlorodihydrofluorescein diacetate (CM-H2DCFDA, Invitrogen). CM-H2DCFDA stock (1 m) was prepared in DMSO. MRC-5 cells on coverslips were washed twice with PBS, incubated in phenol red-free RPMI medium containing 5 μm CM-H2DCFDA for 15 min, washed twice with PBS, and then incubated in phenol red-free RPMI medium for an additional 15 min in an incubator. Cells were then imaged under a Zeiss AxioObserver 100 fluorescence microscope for image acquisition using the FITC 488-nm excitation spectra setting. Immunofluorescence and immunoblotting analyses were performed as described previously (34Wu M. Kang M.M. Schoene N.W. Cheng W.H. Selenium compounds activate early barriers of tumorigenesis.J. Biol. Chem. 2010; 285: 12055-12062Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Qi Y. Schoene N.W. Lartey F.M. Cheng W.H. Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.J. Biol. Chem. 2010; 285: 33010-33017Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). Focus positive cells were defined as those containing at least five foci within the nucleus (34Wu M. Kang M.M. Schoene N.W. Cheng W.H. Selenium compounds activate early barriers of tumorigenesis.J. Biol. Chem. 2010; 285: 12055-12062Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar, 35Qi Y. Schoene N.W. Lartey F.M. Cheng W.H. Selenium compounds activate ATM-dependent DNA damage response via the mismatch repair protein hMLH1 in colorectal cancer cells.J. Biol. Chem. 2010; 285: 33010-33017Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 37Maude S.L. Enders G.H. Cdk inhibition in human cells compromises chk1 function and activates a DNA damage response.Cancer Res. 2005; 65: 780-786PubMed Google Scholar). Briefly, permeabilized cells were incubated overnight at 4 °C with antibodies against phospho-H2AX on Ser-139 (γH2AX, 1:200; Abcam, Cambridge, MA), phospho-ATM on Ser-1981 (pATM Ser-1981, 1:500; Rockland, Gilbertsville, PA), H2AX (1:500; Abcam), and ATM (1:500; Epitomics, Burlingame, CA). γH2AX and pATM Ser-1981 are well-defined markers for DNA breaks and ATM pathway activation, respectively (38Löbrich M. Shibata A. Beucher A. Fisher A. Ensminger M. Goodarzi A.A. Barton O. Jeggo P.A. gammaH2AX foci analysis for monitoring DNA double-strand break repair: strengths, limitations and optimization.Cell Cycle. 2010; 9: 662-669Crossref PubMed Scopus (498) Google Scholar, 39Bakkenist C.J. Kastan M.B. DNA damage activates ATM through intermolecular autophosphorylation and dimer dissociation.Nature. 2003; 421: 499-506Crossref PubMed Scopus (2684) Google Scholar). Six pictures were randomly taken from each slide. Nuclear fraction was prepared by using a Nuclear and Cytoplasmic Extraction Kit (G-Bioscience, MO), separated by SDS-PAGE, and transferred onto PVDF membranes. Blots were incubated with antibodies against phospho-Nrf2 on Ser-40 (pNrf2, 1:1000; Epitomics), Nrf2 (1:1000; Santa Cruz Biotechnology), and lamin B (1:1000; Santa Cruz Biotechnology), followed by incubation with HRP-conjugated secondary antibodies and chemiluminescent reagents (Super Signal, Pierce) for signal acquisition. All experiments were performed in duplicate and a minimum of three times. The activity of senescence-associated β-galactosidase (SA-β-gal), a senescence marker undetectable in actively proliferating cells (10Dimri G.P. Lee X. Basile G. Acosta M. Scott G. Roskelley C. Medrano E.E. Linskens M. Rubelj I. Pereira-Smith O. A biomarker that identifies senescent human cells in culture and in aging skin in vivo.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 9363-9367Crossref PubMed Scopus (5707) Google Scholar), was determined using a Senescence Detection Kit (BioVision, San Francisco, CA) according to manufacturer's instruction. MRC-5 cells were seeded onto 12-well plates (104 cells/well) in the presence or absence of H2O2 (20 μm) in a 3% or a 20% O2 incubator. Images were captured under a light microscope for quantification. Autofluorescence is another index of aging in cultured human diploid fibroblasts (40Wilhelm J. Vytásek R. Ostádalová I. Vajner L. Evaluation of different methods detecting intracellular generation of free radicals.Mol. Cell Biochem. 2009; 328: 167-176Crossref PubMed Scopus (26) Google Scholar). Passage 4 SelH and scrambled shRNA MRC-5 cells were subjected to Zeiss AxioObserver 100 fluorescence microscope at 200× magnification. The fluorescence signal was acquired by using filter cube set 49 (excitation, 365 nm; filter, 395 nm; emission, 445 nm), and the intensity of autofluorescence was analyzed by Axiovision software. After being seeded (750 cells/6-cm dish) for 24 h, SelH and scrambled shRNA HeLa and HCT116+hMLH1 cells were incubated with a gradient concentration of DNA-damaging agents, including hydroxyurea, neocarzinostatin, camptothecin, paraquat, and H2O2 (Fisher Scientific) for 24 h. Then, the drug-containing medium was replaced by a complete medium and cultured for additional 7 days. Cells were washed with 1× PBS, fixed in 90% methanol, and stained by 0.5% crystal violet (Alfa Aesar, MA) in 25% methanol. A viable colony is defined as one composing of more than 50 cells. A mixture of oxidative and broken DNA damage was generated in live cell nuclei by laser-induced microirradiation using a pulsed nitrogen laser as previously described (41Uematsu N. Weterings E. Yano K. Morotomi-Yano K. Jakob B. Taucher-Scholz G. Mari P.O. van Gent D.C. Chen B.P. Chen D.J. Autophosphorylation of DNA-PKCS regulates its dynamics at DNA double-strand breaks.J. Cell Biol. 2007; 177: 219-229Crossref PubMed Scopus (309) Google Scholar). SelH-GFP (24Novoselov S.V. Kryukov G.V. Xu X.M. Carlson B.A. Hatfield D.L. Gladyshev V.N. Selenoprotein H is a nucleolar thioredoxin-like protein with a unique expression pattern.J. Biol. Chem. 2007; 282: 11960-11968Abstract Full Text Full Text PDF PubMed Scopus (96) Google Scholar) and proliferating cell nuclear antigen (PCNA)-DsRed plasmids were transiently transfected into MRC-5 cells. The laser system was coupled to a Zeiss Axiovert microscope for live cell, time lapse image capture. Total intracellular GSH was estimated using monochlorobimane (mBCL), which formed fluorescent adducts with GSH (42Sebastià J. Cristòfol R. Martín M. Rodríguez-Farré E. Sanfeliu C. Evaluation of fluorescent dyes for measuring intracellular glutathione content in primary cultures of human neurons and neuroblastoma SH-SY5Y.Cytometry A. 2003; 51: 16-25Crossref PubMed Scopus (103) Google Scholar, 43Franco R. Cidlowski J.A. SLCO/OATP-like transport of glutathione in FasL-induced apoptosis: glutathione efflux is coupled to an organic anion exchange and is necessary for the progression of the execution phase of apoptosis.J. Biol. Chem. 2006; 281: 29542-29557Abstract Full Text Full Text PDF PubMed Scopus (97) Google Scholar). Adherent cells were collected and preloaded with mBCl (40 μm) in PBS for 10 min, followed by incubation with propyl iodide permeable only to dead cells. The stained cells were applied to FACSCanto II flow cytometer (BD Bioscience, CA) immediately to determine the content of GSH per 10,000 live cells. Apoptotic cells were determined by a Mitocapture kit (Biovision) on the basis of the disruption of mitochondrial membrane potential. The Mitocapture reagent accumulated in the mitochondria and showed red fluorescence only in live cells, but could not aggregate and showed green fluorescence in apoptotic cells. Cells on coverslips were incubated with 1:1000 Mitocapture reagent (diluted in pre-warmed incubation buffer) in an incubator for 15 min, and washed three times with incubation buffer. The stained cells were subjected to fluorescent microscopic analyses of GFP and DsRed signals. Eight pictures were randomly taken from each slide. Strikingly, SelH shRNA MRC-5 cells barely proliferated, accumulated non-dividing large and flat cells, and showed completely stalled growth by passage 4 or 36 days after clonal selection, whereas scrambled shRNA MRC-5 cells proliferated exponentially (Fig. 1, A and B). These results indicate that SelH deficiency restricts replicative lifespan in human diploid fibroblasts. Old or senescent cells in culture have previously been shown to exhibit elevated autofluorescence (40Wilhelm J. Vytásek R. Ostádalová I. Vajner L. Evaluation of different methods detecting intracellular generation of free radicals.Mol. Cell Biochem. 2009; 328: 167-176Crossref PubMed Scopus (26) Google Scholar, 44Kuilman T. Michaloglou C. Mooi W.J. Peeper D.S. The essence of senescence.Genes Dev. 2010; 24: 2463-2479Crossref PubMed Scopus (1434) Google Scholar). Consistent with these observations, microscopic analyses of passage 4, non-proliferating SelH shRNA MRC-5 cells showed 7-fold greater autofluorescence signal as compared with scrambled shRNA cells (Fig. 1B). Further microscopic analyses of CM-H2DCFDA fluorescence demonstrated that level of intracellular ROS was 2-fold greater in SelH than in scrambled shRNA MRC-5 cells at passage 4 when cultured under 20% O2 (Fig. 1C). Overall, these results implicate ROS in the slow proliferation and the formation of senescence-like phenotypes in SelH shRNA MRC-5 cells. Because fibroblasts cultured in a typical 20% O2 incubator is believed to be under chronic oxygen tension and can accelerate replicative senescence (45Parrinello S. Samper E. Krtolica A. Goldstein J. Melov S. Campisi J. Oxygen sensitivity severely limits the replicative lifespan of murine fibroblasts.Nat. Cell Biol. 2003; 5: 741-747Crossref PubMed Scopus (880) Google Scholar, 46Poulios E. Trougakos I.P. Chondrogianni N. Gonos E.S. Exposure of human diploid fibroblasts to hypoxia extends proliferative life span.Ann. N.Y. Acad. Sci. 2007; 1119: 9-19Crossref PubMed Scopus (22) Google Scholar), features of senescence in MRC-5 cells were also assessed under a 3% O2 (physiological level) culture condition. While SA-β-gal expression was significantly greater (p < 0.05) in SelH than in scrambled shRNA MRC-5 cells being cultured either in a 3% or a 20% O2 incubator for 7 days (Fig. 1D), the extent of which was significantly reduced when cultured under 3% O2 in both SelH and scrambled shRNA MRC-5 cells. Moreover, the complete growth inhibition of SelH shRNA MRC-5 cells maintained under 20% O2 for 5 weeks could be partially rescued when grown in a 3% O2 incubator, although SelH shRNA MRC-5 cells remained to proliferate poorly (∼300-fold slower) as compared with scrambled shRNA MRC-5 cells (Fig. 1, A and E). Similarly, scrambled shRNA MRC-5 cells proliferated 55-fold faster in a 3% than in a 20% O2 incubator. Altogether, these results suggest that SelH is required for cellular proliferation and the suppression of replicative senescence in a manner depending on ROS in human diploid fibroblasts. Senescence in human diploid fibroblasts is associated with persistent broken and oxidative DNA damage (47Campisi J. d'Adda di Fagagna F. Cellular senescence: when bad things happen to good cells.Nat. Rev. Mol. Cell Biol. 2007; 8: 729-740Crossref PubMed Scopus (2977) Google Scholar, 48Chen Q. Fischer A. Reagan J.D. Yan L.J. Ames B.N. Oxidative DNA damage and senescence of human diploid fibroblast cells.Proc. Natl. Acad. Sci. U.S.A. 1995; 92: 4337-4341Crossref PubMed Scopus (573) Google Scholar), both of which can result in ATM pathway activation (11Lee J.H. Paull T.T. ATM activation by DNA double-strand breaks through the Mre11-Rad50-Nbs1 complex.Science. 2005; 308: 551-554Crossref PubMed Scopus (1073) Google Scholar, 12Guo Z. Kozlov S. Lavin M.F. Person M.D. Paull T.T. ATM activation by oxidative stress.Science. 2010; 330: 517-521Crossref PubMed Scopus (797) Google Scholar, 49Tanaka T. Halicka H.D. Huang X. Traganos F. Darzynkiewicz Z. Constitutive histone H2AX phosphorylation and ATM activation, the reporters of DNA damage by endogenous oxidants.Cell Cycle. 2006; 5: 1940-1945Crossref PubMed Scopus (179) Google Scholar). Thus, SelH and scrambled shRNA MRC-5 cells were treated with H2O2 (20 μm) for 1 day, followed by 0–5 days recovery. The percent γH2AX and pATM Ser-1981 positive cells were greater (p < 0.05) in SelH than in scrambled shRNA cells before and 1 day after H2O2 treatment (Fig. 2, A and B). During the course of 5-day recovery, the abundance of γH2AX and pATM Ser-1981 positive cells subsided in the scrambled shRNA cells whereas thes" @default.
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• W2004828905 date "2014-12-01" @default.
• W2004828905 modified "2023-10-14" @default.
• W2004828905 title "Selenoprotein H Suppresses Cellular Senescence through Genome Maintenance and Redox Regulation" @default.
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• W2004828905 doi "https://doi.org/10.1074/jbc.m114.611970" @default.
• W2004828905 hasPubMedCentralId "https://www.ncbi.nlm.nih.gov/pmc/articles/4256366" @default.
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