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• W2127165936 abstract "Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc -/- cells in response to oxidative stress. Our data indicate that Fancc -/- cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc -/- cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc -/- cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H2O2-treated Fancc -/- cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H2O2-induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc -/- hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation. Fanconi anemia is a genetic disorder characterized by bone marrow failure. Significant evidence supports enhanced apoptosis of hematopoietic stem/progenitor cells as a critical factor in the pathogenesis of bone marrow failure in Fanconi anemia. However, the molecular mechanism(s) responsible for the apoptotic phenotype are incompletely understood. Here, we tested whether alterations in the activation of a redox-dependent pathway may participate in the pro-apoptotic phenotype of primary Fancc -/- cells in response to oxidative stress. Our data indicate that Fancc -/- cells are highly sensitive to oxidant stimuli and undergo enhanced oxidant-mediated apoptosis compared with wild type controls. In addition, antioxidants preferentially enhanced the survival of Fancc -/- cells. Because oxidative stress activates the redox-dependent ASK1 pathway, we assessed whether Fancc -/- cells exhibited increased oxidant-induced ASK1 activation. Our results revealed ASK1 hyperactivation in H2O2-treated Fancc -/- cells. Furthermore, using small interfering RNAs to decrease ASK1 expression and a dominant negative ASK1 mutant to inhibit ASK1 kinase activity, we determined that H2O2-induced apoptosis was ASK1-dependent. Collectively, these data argue that the predisposition of Fancc -/- hematopoietic stem/progenitor cells to apoptosis is mediated in part through altered redox regulation and ASK1 hyperactivation. Fanconi anemia (FA) 1The abbreviations used are: FA, Fanconi anemia; BM, bone marrow; TNF, tumor necrosis factor; CPR, cytochrome P-450 reductase; GST, glutathione S-transferase; ASK1, apoptosis signal-regulating kinase 1; WT, wild type; MEF, murine embryo fibroblast; SeMet, selenomethionine; NAC, N-acetylcysteine; EGFP, enhanced green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; DAPI, 4′,6-diamidino-2′-phenylindole-dihydrochrolide; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling. 1The abbreviations used are: FA, Fanconi anemia; BM, bone marrow; TNF, tumor necrosis factor; CPR, cytochrome P-450 reductase; GST, glutathione S-transferase; ASK1, apoptosis signal-regulating kinase 1; WT, wild type; MEF, murine embryo fibroblast; SeMet, selenomethionine; NAC, N-acetylcysteine; EGFP, enhanced green fluorescent protein; MOPS, 4-morpholinepropanesulfonic acid; siRNA, small interfering RNA; DAPI, 4′,6-diamidino-2′-phenylindole-dihydrochrolide; TUNEL, terminal deoxynucleotidyltransferase-mediated nick-end labeling. is a heterogeneous bone marrow (BM) failure syndrome with cellular abnormalities that include chromosomal instability, increased apoptosis, and cell cycle control defects (1Alter B. Young N. Nathan D. Oski F. Hematology of Infancy and Childhood. 5th Ed. 1. W. B. Saunders, Philadelphia1998: 27-335Google Scholar, 2Liu J. Young N.S. Bone Marrow Failure Syndromes. W. B. Saunders, Philadelphia2000: 47-68Google Scholar, 3Joenje H. Patel K.J. Nat. Rev. Genet. 2001; 2: 446-457Crossref PubMed Scopus (504) Google Scholar, 4D'Andrea A.D. Grompe M. Nat. Rev. Cancer. 2003; 3: 23-34Crossref PubMed Scopus (669) Google Scholar, 5Fagerlie S. Lensch M.W. Pang Q. Bagby Jr., G.C. Exp. Hematol. 2001; 29: 1371-1381Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). The diversity of clinical presentation in children with FA is related, in part, to the existence of multiple complementation types, with eight FA complementation group cDNAs being identified thus far (FANCA, FANCC, FANCD1/BRCA2, FANCD2, FANCE, FANCF, FANCG, and FANCL) (6The Fanconi Anaemia/Breast Cancer Consortium Nat. Genet. 1996; 14: 324-328Crossref PubMed Scopus (261) Google Scholar, 7Lo Ten Foe J. Rooimans M. Bosnoyan-Collins L. Alon N. Wijker M. Parker L. Lightfoot J. Carreau M. Callen D. Savoia A. Cheng N. van Berkel C. Strunk M. Gille J. Pals G. Kruyt F. Pronk J. Arwert F. Buchwald M. Joenje H. Nat. Genet. 1996; 14: 320-323Crossref PubMed Scopus (301) Google Scholar, 8Strathdee C. Gavish H. Shannon W. Buchwald M. Nature. 1992; 356: 763-767Crossref PubMed Scopus (535) Google Scholar, 9Timmers C. Taniguchi T. Hejna J. Reifsteck C. Lucas L. Bruun D. Thayer M. Cox B. Olson S. D'Andrea A.D. Moses R. Grompe M. Mol. Cell. 2001; 7: 241-248Abstract Full Text Full Text PDF PubMed Scopus (330) Google Scholar, 10de Winter J. Waisfisz Q. Rooimans M. van Berkel C. Bosnoyan-Collins L. Alon N. Carreau M. Bender O. Demuth I. Schindler D. Pronk J. Arwert F. Hoehn H. Digweed M. Buchwald M. Joenje H. Nat. Genet. 1998; 20: 281-283Crossref PubMed Scopus (283) Google Scholar, 11de Winter J.P. Leveille F. van Berkel M. Rooimans M. van der Weel L. Sheltenpool J. Demuth I. Morgan N. Alon N. Bosnoyan-Collins L. Lightfoot J. Leegwater P.A. Waisfisz Q. Komatsu K. Arwert F. Pronk J. Mathew C. Digweed M. Buchwald M. Joenje H. Am. J. Hum. Genet. 2000; 67: 1306-1308Abstract Full Text Full Text PDF PubMed Scopus (190) Google Scholar, 12de Winter J.P. Rooimans M.A. van Der Weel L. van Berkel C.G. Alon N. Bosnoyan-Collins L. de Groot J. Zhi Y. Waisfisz Q. Pronk J.C. Arwert F. Mathew C.G. Scheper R.J. Hoatlin M.E. Buchwald M. Joenje H. Nat. Genet. 2000; 24: 15-161Crossref PubMed Scopus (236) Google Scholar, 13Howlett N.G. Taniguchi T. Olson S. Cox B. Waisfisz Q. De Die-Smulders C. Persky N. Grompe M. Joenje H. Pals G. Ikeda H. Fox E.A. D'Andrea A.D. Science. 2002; 297: 606-609Crossref PubMed Scopus (963) Google Scholar, 14Meetei A.R. De Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. Van De Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar). Despite some clinical variability between individuals with specific FA gene mutations (15Gillio A. Verlander P. Batish S. Giampietro P. Auerbach A. Blood. 1997; 90: 105-110Crossref PubMed Google Scholar, 16Faivre L. Guardiola P. Lewis C. Dokal I. Ebell W. Zatterale A. Altay C. Poole J. Stones D. Kwee M.L. van Weel-Sipman M. Havenga C. Morgan N. de Winter J. Digweed M. Savoia A. Pronk J. de Ravel T. Jansen S. Joenje H. Gluckman E. Mathew C.G. Blood. 2000; 96: 4064-4070PubMed Google Scholar), the major cause of mortality in all FA complementation types is BM failure (1Alter B. Young N. Nathan D. Oski F. Hematology of Infancy and Childhood. 5th Ed. 1. W. B. Saunders, Philadelphia1998: 27-335Google Scholar, 2Liu J. Young N.S. Bone Marrow Failure Syndromes. W. B. Saunders, Philadelphia2000: 47-68Google Scholar, 5Fagerlie S. Lensch M.W. Pang Q. Bagby Jr., G.C. Exp. Hematol. 2001; 29: 1371-1381Abstract Full Text Full Text PDF PubMed Scopus (29) Google Scholar). These studies suggest that apoptotic loss of hematopoietic stem/progenitor cells has a key pathogenetic role in this disorder. Thus, understanding molecular mechanisms involved in the predisposition of FA cells to apoptosis is of critical importance to improve current treatment approaches for children with FA. Numerous studies show that FANCA, FANCC, FANCE, FANCF, and FANCG interact in a multimeric nuclear protein complex (17Kupfer G. Naf D. Suliman A. Pulsipher M. D'Andrea A. Nat. Genet. 1997; 17: 487-490Crossref PubMed Scopus (159) Google Scholar, 18Garcia-Higuera I. Kuang Y. Naf D. Wasik J. D'Andrea A.D. Mol. Cell Biol. 1999; 19: 4866-4873Crossref PubMed Scopus (199) Google Scholar, 19de Winter J.P. van der Weel L. de Groot J. Stone S. Waisfisz Q. Arwert F. Scheper R.J.F.A.E.,K. Hoatlin M.E. Joenje H. Hum. Mol. Genet. 2000; 9: 2665-2674Crossref PubMed Scopus (172) Google Scholar, 20Pace P. Johnson M. Tan W.M. Mosedale G. Sng C. Hoatlin M. de Winter J. Joenje H. Gergely F. Patel K.J. EMBO J. 2002; 21: 3414-3423Crossref PubMed Scopus (138) Google Scholar, 21Medhurst A.L. Huber P.A. Waisfisz Q. de Winter J.P. Mathew C.G. Hum. Mol. Genet. 2001; 10: 423-429Crossref PubMed Scopus (140) Google Scholar, 22Meetei A.R. Sechi S. Wallisch M. Yang D. Young M.K. Joenje H. Hoatlin M.E. Wang W. Mol. Cell Biol. 2003; 23: 3417-3426Crossref PubMed Scopus (292) Google Scholar, 23Gordon S.M. Buchwald M. Blood. 2003; 102: 136-141Crossref PubMed Scopus (52) Google Scholar), the formation of which is required for FANCL to monoubiquitinate FANCD2 (14Meetei A.R. De Winter J.P. Medhurst A.L. Wallisch M. Waisfisz Q. Van De Vrugt H.J. Oostra A.B. Yan Z. Ling C. Bishop C.E. Hoatlin M.E. Joenje H. Wang W. Nat. Genet. 2003; 35: 165-170Crossref PubMed Scopus (471) Google Scholar), a post-translational modification that signals relocalization of FANCD2 into nuclear foci containing BRCA1 (24Garcia-Higuera I. Taniguchi T. Ganesan S. Meyn M.S. Timmers C. Hejna J. Grompe M. D'Andrea A.D. Mol. Cell. 2001; 7: 249-262Abstract Full Text Full Text PDF PubMed Scopus (1022) Google Scholar). However, the majority of FANCC resides in the cytoplasm (25Youssoufian H. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 7975-7979Crossref PubMed Scopus (112) Google Scholar, 26Heinrich M. Silvey K. Stone S. Zigler A. Griffith D. Montalto M. Chai L. Zhi Y. Hoatlin M. Blood. 2000; 95: 3970-3977Crossref PubMed Google Scholar), and cytoplasmic localization is required for protection against genotoxin-induced apoptosis (27Youssoufian H. J. Clin. Invest. 1996; 97: 2003-2010Crossref PubMed Scopus (58) Google Scholar). Interestingly, cytoplasmic FANCC interacts with heat shock protein 70 (HSP70) to protect cells from interferon-γ/TNF-α-induced apoptosis, yet FANCC-HSP70 interaction is dispensable for protection against genotoxic stress (28Pang Q. Keeble W. Christianson T.A. Faulkner G.R. Bagby G.C. EMBO J. 2001; 20: 4478-4489Crossref PubMed Scopus (115) Google Scholar, 29Pang Q. Christianson T.A. Keeble W. Diaz J. Faulkner G.R. Reifsteck C. Olson S. Bagby G.C. Blood. 2001; 98: 1392-1401Crossref PubMed Scopus (69) Google Scholar, 30Pang Q. Christianson T.A. Keeble W. Koretsky T. Bagby G.C. J. Biol. Chem. 2002; 277: 49638-49643Abstract Full Text Full Text PDF PubMed Scopus (84) Google Scholar). Together these data support an additional cytoplasmic role for FANCC in suppressing apoptosis. Participation of FANCC in redox metabolism has been proposed previously and is supported by functional interactions with cytochrome P-450 reductase (CPR) (31Kruyt F. Hoshino T. Liu J. Joseph P. Jaiswal A. Youssoufian H. Blood. 1998; 92: 3050-3056Crossref PubMed Google Scholar) and glutathione S-transferase P1 (GSTP1) (32Cumming R.C. Lightfoot J. Beard K. Youssoufian H. O'Brien P.J. Buchwald M. Nat. Med. 2001; 7: 814-820Crossref PubMed Scopus (207) Google Scholar). Evidence of oxygen sensitivity was first provided by Joenje et al. (34Joenje H. Youssoufian H. Kruyt F. dos Santos C. Wevrick R. Buchwald M. Blood Cells Mol. Dis. 1995; 21: 182-191Crossref PubMed Scopus (14) Google Scholar), who demonstrated that the chromosomal instability of primary FA cells could be reduced if grown at lowered oxygen tension (33Joenje H. Arwert F. Eriksson A. de Koning H. Oostra A. Nature. 1981; 290: 142-143Crossref PubMed Scopus (247) Google Scholar). Several conflicting studies have been reported since this original description, although most analyses were conducted in immortalized cell lines or in cells with unidentified FA complementation type and/or mutation (34Joenje H. Youssoufian H. Kruyt F. dos Santos C. Wevrick R. Buchwald M. Blood Cells Mol. Dis. 1995; 21: 182-191Crossref PubMed Scopus (14) Google Scholar, 35Saito H. Hammond A. Moses R. Mutat. Res. 1993; 294: 255-262Crossref PubMed Scopus (58) Google Scholar, 36Kupfer G. D'Andrea A. Blood. 1996; 88: 1019-1025Crossref PubMed Google Scholar). Now with the availability of murine models, the issue of whether FA cells have altered redox regulation is being readdressed in primary cells. Using mice deficient in the murine FANCC homologue (Fancc), Hadjur et al. (37Hadjur S. Ung K. Wadsworth L. Dimmick J. Rajcan-Separovic E. Scott R.W. Buchwald M. Jirik F.R. Blood. 2001; 98: 1003-1011Crossref PubMed Scopus (68) Google Scholar) showed that mice mutant at both the Fancc and superoxide dismutase 1 (SOD1) loci exhibit severe defects in hematopoiesis, including histological evidence of BM hypoplasia, an observation not detected in singly mutant mice. Although these data provide strong genetic evidence that Fancc -/- cells are hypersensitive to endogenously generated oxidants, it is unknown whether the molecular mechanism responsible for this hyper-sensitive response is due to altered redox signaling. Redox signaling has a critical role in controlling multiple complex cellular processes including apoptosis, proliferation, senescence, and differentiation (38Adler V. Yin Z. Tew K.D. Ronai Z. Oncogene. 1999; 18: 6104-6111Crossref PubMed Scopus (592) Google Scholar, 39Lander H.M. FASEB J. 1997; 11: 118-124Crossref PubMed Scopus (822) Google Scholar, 40Lander H.M. Ogiste J.S. Teng K.K. Novogrodsky A. J. Biol. Chem. 1995; 270: 21195-21198Abstract Full Text Full Text PDF PubMed Scopus (370) Google Scholar, 41Sundaresan M. Yu Z.X. Ferrans V.J. Irani K. Finkel T. Science. 1995; 270: 296-299Crossref PubMed Scopus (2312) Google Scholar, 42Schafer F.Q. Buettner G.R. Free Radic. Biol. Med. 2001; 30: 1191-1212Crossref PubMed Scopus (3647) Google Scholar, 43Rhee S.G. Bae Y.S. Lee S.R. Kwon J. Science's STKE. 2000; http://stke.sciencemag.org/cgi/content/full/sigtrans;2000/53/pe1PubMed Google Scholar, 44Powis G. Gasdaska J.R. Baker A. Adv. Pharmacol. 1997; 38: 329-359Crossref PubMed Scopus (114) Google Scholar). This highly conserved regulatory process involves maintenance of the intracellular environment in an overall reduced state. Cellular oxidative stress results in the oxidation of key cysteine residues on redox-sensitive proteins, a post-translational modification that affects intracellular signaling pathways in a fashion similar to phosphorylation. A notable example of redox apoptotic signaling involves the serine-threonine kinase apoptosis signal-regulating kinase 1 (ASK1). In the normal reducing environment of a cell, ASK1 activity is inhibited via binding to thioredoxin, glutaredoxin, and glutathione S-transferases (45Cho S.G. Lee Y.H. Park H.S. Ryoo K. Kang K.W. Park J. Eom S.J. Kim M.J. Chang T.S. Choi S.Y. Shim J. Kim Y. Dong M.S. Lee M.J. Kim S.G. Ichijo H. Choi E.J. J. Biol. Chem. 2001; 276: 12749-12755Abstract Full Text Full Text PDF PubMed Scopus (347) Google Scholar, 46Saitoh M. Nishitoh H. Fujii M. 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After direct or indirect oxidant stress (i.e. H2O2, TNF-α, glucose/serum deprivation, and heat shock), these proteins are oxidized forming intramolecular disulfide bonds, which result in a conformational change and dissociation from ASK1. Unbound ASK1 is then available to autophosphorylate and subsequently phosphorylate downstream kinases, initiating an apoptotic program. To extend our understanding of oxidant hypersensitivity in FA, we investigated whether primary Fancc -/- cells exhibit alterations in the redox-dependent ASK1 apoptotic pathway. Our data demonstrate that Fancc -/- cells exhibit ASK1 hyperactivation after H2O2 treatment. In addition, we show that enhanced H2O2-induced apoptosis in Fancc -/- cells is ASK1-dependent and that pretreatment with antioxidants preferentially protects Fancc -/- cells from apoptosis induced by H2O2 as compared with controls. Collectively, these data argue that the predisposition of primary Fancc -/- cells to oxidant-induced apoptosis is mediated through hyperactivation of a redox-dependent apoptotic signaling pathway. Mice—Fancc +/- mice in a C57Bl/6J genetic background were bred to generate Fancc -/- and wild type (WT) mice for hematopoietic progenitor assays and timed embryos for murine embryo fibroblast (MEF) cell lines as previously described (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar, 53Freie B. Li X. Ciccone S.L.M. Nawa K. Cooper S. Vogelweid C. Schantz L. Haneline L.S. Orazi A. Broxmeyer H.E. Lee S.-H. Clapp D.W. Blood. 2003; 102: 4146-4152Crossref PubMed Scopus (54) Google Scholar). All of the studies were approved by the Indiana University Laboratory Animal Research Center. Hematopoietic Progenitor Assays—WT and Fancc -/- BM low density mononuclear and ckit+lin- cells were prepared as previously described (54Haneline L.S. Gobbett T.A. Ramani R. Carreau M. Buchwald M. Yoder M.C. Clapp D.W. Blood. 1999; 94: 1-8Crossref PubMed Google Scholar, 55Haneline L.S. Broxmeyer H.E. Cooper S. Hangoc G. Carreau M. Buchwald M. Clapp D.W. Blood. 1998; 91: 4092-4098Crossref PubMed Google Scholar). Cells from WT and Fancc -/- mice were resus-pended in Iscove's modified Dulbecco's medium (Invitrogen) supplemented with 20% fetal calf serum (Biowhittaker, Walkersville, MD) and then exposed to H2O2 (Sigma) for 1 h. After oxidant treatment, the cells were washed and plated in clonogenic assays as described previously (55Haneline L.S. Broxmeyer H.E. Cooper S. Hangoc G. Carreau M. Buchwald M. Clapp D.W. Blood. 1998; 91: 4092-4098Crossref PubMed Google Scholar). For hyperoxia exposure, low density mononuclear cells were placed in an airtight chamber before infusing with a gas mixture containing 50% O2, 5% CO2, and 45% N2 (Praxair, Indianapolis, IN). The chamber was then incubated for 4 or 16 h at 37 °C before the cells were harvested for clonogenic assays. An O2 analyzer was used to measure the O2 concentration before and after each incubation period (50 + 3%) to ensure an airtight culture system. Control cultures were incubated at 21% O2 for 4 or 16 h. MEF Survival Assays—MEFs were maintained as previously described (53Freie B. Li X. Ciccone S.L.M. Nawa K. Cooper S. Vogelweid C. Schantz L. Haneline L.S. Orazi A. Broxmeyer H.E. Lee S.-H. Clapp D.W. Blood. 2003; 102: 4146-4152Crossref PubMed Scopus (54) Google Scholar). All of the studies were conducted in at least two or three different MEF cell lines/genotype, and only MEFs that were less than passage 5 were utilized. To assess H2O2 sensitivity, WT and Fancc -/- MEFs were cultured with H2O2 for 24 h before assessing viability by trypan blue exclusion. In some experiments, MEFs were pretreated with 20 μm selenomethionine (SeMet; Sigma) overnight or 4 mmN-acetylcysteine (NAC; Sigma) for 1 h prior to culturing with H2O2. To evaluate apoptosis, MEFs were treated with 100 μm H2O2 for 4-6 h and analyzed by the terminal deoxynucleotidyltransferase-mediated nick-end labeling (TUNEL) assay as previously described (55Haneline L.S. Broxmeyer H.E. Cooper S. Hangoc G. Carreau M. Buchwald M. Clapp D.W. Blood. 1998; 91: 4092-4098Crossref PubMed Google Scholar, 56Freie B. Li X. Ciccone S.L. Nawa K. Cooper S. Vogelweid C. Schantz L. Haneline L.S. Orazi A. Broxmeyer H.E. Lee S.-H. Clapp D.W. Blood. 2003; 102: 4146-4152Crossref PubMed Scopus (56) Google Scholar). Retroviral Constructs and Transduction—PG13 retroviral packaging cells containing the FANCC mutants (FANCC-E251A and FANCC-del322G) in the pLXSN backbone were generously provided by Dr. Grover C. Bagby, Jr. (Oregon Health Sciences, Portland, OR) (29Pang Q. Christianson T.A. Keeble W. Diaz J. Faulkner G.R. Reifsteck C. Olson S. Bagby G.C. Blood. 2001; 98: 1392-1401Crossref PubMed Scopus (69) Google Scholar). Retroviral supernatants were harvested and utilized to transduce GP+E86 retroviral packaging cells as previously described (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar) to pseudotype viral particles with an ecotropic envelope. The MFG-FAC retrovirus encoding the FANCC cDNA was used as a control, which previously was shown to correct mitomycin C sensitivity of Fancc -/- cells to WT levels (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar). The dominant negative ASK1 cDNA (ASK1-K709M) (57Ichijo H. Nishida E. Irie K. ten Dijke P. Saitoh M. Moriguchi T. Takagi M. Matsumoto K. Miyazono K. Gotoh Y. Science. 1997; 275: 90-94Crossref PubMed Scopus (2022) Google Scholar) was generously provided by Dr. Hidenori Ichijo (University of Tokyo, Tokyo, Japan) in a pcDNA plasmid. The ASK1-K709M cDNA was subcloned into the NotI site of the bicistronic retroviral plasmid MIEG3 (58Tao W. Filippi M.D. Bailey J.R. Atkinson S.J. Connors B. Evan A. Williams D.A. Blood. 2002; 100: 1679-1688Crossref PubMed Google Scholar), which is 5′ to an internal ribosomal entry site-enhanced green fluorescent protein (EGFP) cassette. A GP+E86 packaging cell line was developed for MIEG3 and MIEG3-ASK1-K709M as previously described (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar). Retroviral supernatants were collected, filtered, and stored at -80 °C until utilized for transduction of MEFs. Early passage MEFs (P0-P1) were transduced with retroviral supernatants four times over 2 consecutive days in the presence of polybrene as previously described (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar, 53Freie B. Li X. Ciccone S.L.M. Nawa K. Cooper S. Vogelweid C. Schantz L. Haneline L.S. Orazi A. Broxmeyer H.E. Lee S.-H. Clapp D.W. Blood. 2003; 102: 4146-4152Crossref PubMed Scopus (54) Google Scholar). ASK1 in Vitro Kinase Assay—ASK1 kinase activity was determined by depriving MEFs of serum for 4 h followed by treatment with 100 μm H2O2 for 5 min. MEFs were then washed twice with cold phosphate-buffered saline containing 1 mm sodium orthovanadate and lysed in nonionic lysis buffer. The protein concentrations were determined using the BCA assay (Pierce). The ASK1 immunoprecipitations were conducted using protein A Sepharose beads (Amersham Biosciences) and anti-ASK1 antibody (Cell Signaling, Beverly, MA). Immunobeads were subjected to an in vitro kinase reaction using either myelin basic protein (Sigma) or MKK4 (Upstate Biotechnologies, Inc.) as substrates for ASK1. The kinase mixtures contained 20 mm MgCl2, 0.1 m sodium orthovanadate, 1 m dithiothreitol, 30 mm β glycerol phosphate, 5 mm EGTA, 20 mm MOPS, 1 μm ATP, and 10 μg of substrate/sample before adding 2.5 μCi of [γ-32P]ATP/sample. The kinase reaction buffer was added to each sample and incubated at 30 °C for 30 min. The reactions were terminated by the addition of sample buffer. The protein samples were separated on a 12% SDS-PAGE gel (Invitrogen), transferred to a nitrocellulose membrane, and subjected to autoradiography. Western Blotting—Equivalent amounts of protein (200-500 μg) were separated on a 12% SDS-PAGE gel and transferred onto a nitrocellulose membrane. For immunodetection of FANCC mutants, a primary rabbit anti-FANCC antibody, previously generated by our laboratory (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar), and a secondary anti-rabbit horseradish peroxidase antibody (Amersham Biosciences) were used as described (52Haneline L.S. Li X. Ciccone S.L.M. Hong P. Yang Y. Broxmeyer H.E. Lee S.-H. Orazi A. Srour E.F. Clapp D.W. Blood. 2003; 101: 1299-1307Crossref PubMed Scopus (67) Google Scholar) before visualizing by chemiluminescence (Amersham Biosciences). To document equal protein loading, the membrane was stripped and reprobed with β-actin (Sigma). For immunodetection of ASK1, rabbit anti-ASK1 antibody (Cell Signaling) was used at a 1:200 dilution before incubating with the secondary antibody anti-rabbit horseradish peroxidase (1:2000 dilution). Small Interfering RNA Transfection Protocol—Table I lists the RNA sequences used for these studies. The ASK1 siRNA sequence targeted nucleotides 570-590 of the ASK1 mRNA and was designed according to the manufacturer's recommendations (Dharmicon, Lafayette, CO). Either sense or scrambled oligonucleotides were used as a control for every transfection experiment. WT and Fancc -/- MEFs were cultured in a 6-well tissue culture dish to 30-50% confluency. The RNA oligonucleotides were diluted in Opti-MEM (Invitrogen) to obtain a 250 nm solution per Dharmicon's recommendations. Oligofectamine transfections were conducted exactly per the manufacturer's recommendations (Invitrogen). Following the transfection, 500 μl of Dulbecco's modified Eagle's medium (Invitrogen) containing 30% fetal calf serum was added without removing the transfection mixture. The cells were incubated for 72 h at 37 °C before harvesting for H2O2 cytotoxicity assays and ASK1 Western blotting. Four independent transfections were conducted with similar results.Table IsiRNA sequencesRNA typeSequenceASK1 target5′-CCG CCG UGC UGG ACC GUU UdTdTSense5′-AAA CGG UCC AGC ACG GCG GdTdTScrambled5′-GCG CGC UUU GUA GGA UUC GdTdT Open table in a new tab Statistical Analyses—For all data shown, a Student's t test was conducted to evaluate for differences between treatment groups, and a p value ≤ 0.05 was considered significant. Fancc -/- Hematopoietic Progenitors and MEFs Are Hyper-sensitive to Oxidants—Because FA patients have severe defects in hematopoietic stem/progenitor cell function, we initially tested whether Fancc -/- progenitors were hypersensitive to oxidant stress. To accomplish this aim, WT and Fancc -/- BM low density mononuclear cells were cultured either with H2O2 or in hyperoxic conditions (50% O2) before plating in clonogenic progenitor assays. For studies utilizing H2O2, Fancc -/- progenitors were significantly more sensitive to multiple H2O2 doses as compared with controls (Fig. 1A). In addition, Fancc -/- progenitors exposed to 50% O2 for 4 or 16 h exhibited a marked reduction in colony formation as compared with WT control cultures (Fig. 1B). BM low density mononuclear cells are a heterogeneous cell population that includes a significant proportion of differentiated cells compared with the relatively low frequency of clonogenic progenitor cells (0.01-0.5%). Given our previous observations that Fancc -/- progenitors are exquisitely sensitive to multiple inhibitory cytokines such as interferon-γ and TNF-α (55Haneline L.S. Broxmeyer H.E. Cooper S. Hangoc G. Carreau M. Buchwald M. Clapp D.W. Blood. 1998; 91: 4092-4098Crossref PubMed Google Scholar), together with the knowledge that inflammatory cells such as lymphocytes and granulocytes are major sources of secreted inhibitory cytokines, it was crucial to eliminate these differentiated cells from our culture system. To test whether the observed oxidant hypersensitivity was due to an intrinsic abnormality in Fancc -/- progenitor cells and not secondary to accessory cells present in BM low density mononuclear cell populations, WT and Fancc -/- ckit+lin- cells were purified by fluorescence cytometry, treated with 100 μm H2O2, and plated in colony assays. This phenotypically defined cell population enriches for immature hematopoietic stem and progenitor cells and excludes differentiated progeny cells. Similar to prior studies with low density mononuclear cells, Fancc -/- ckit+lin- cells were hypersensitive to H2O2 (Fig. 1C), supporting an intrinsic hematopoietic progenitor cell defect. Because of the difficulty in obtaining sufficient numbers of primary hematopoietic progenitor cells, we established WT and Fancc -/- MEFs to utilize as a cellular model system for evaluation of alterations in oxidant responsiveness in Fancc -/- cells. Initial studies determined that Fancc -/- MEFs exh" @default.
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• W2127165936 date "2004-04-01" @default.
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• W2127165936 title "Oxidant Hypersensitivity of Fanconi Anemia Type C-deficient Cells Is Dependent on a Redox-regulated Apoptotic Pathway" @default.
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