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Friedreich's Ataxia, No Changes in Mitochondrial Labile Iron in Human Lymphoblasts and Fibroblasts

2004; Elsevier BV; Volume: 280; Issue: 8 Linguagem: Inglês

10.1074/jbc.m408717200

ISSN

1083-351X

Autores

Brigitte Sturm, Ute Bistrich, Matthias Schranzhofer, Joseph P. Sarsero, Ursula Rauen, Barbara Scheiber‐Mojdehkar, Herbert de Groot, Panos Ioannou, Frank Petrat,

Tópico(s)

Parkinson's Disease Mechanisms and Treatments

Resumo

Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with 55Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H2O2 was significantly increased in patient cells, and H2O2 toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2′-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA. Friedreich's ataxia (FRDA) is caused by low expression of frataxin, a small mitochondrial protein. Studies with both yeast and mammals have suggested that decreased frataxin levels lead to elevated intramitochondrial concentrations of labile (chelatable) iron, and consequently to oxidative mitochondrial damage. Here, we used the mitochondrion-selective fluorescent iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA) to determine the mitochondrial chelatable iron of FRDA patient lymphoblast and fibroblast cell lines, in comparison with age- and sex-matched control cells. No alteration in the concentration of mitochondrial chelatable iron could be observed in patient cells, despite strongly decreased frataxin levels. Uptake studies with 55Fe-transferrin and iron loading with ferric ammonium citrate revealed no significant differences in transferrin receptor density and iron responsive protein/iron regulatory element binding activity between patients and controls. However, sensitivity to H2O2 was significantly increased in patient cells, and H2O2 toxicity could be completely inhibited by the ubiquitously distributing iron chelator 2,2′-dipyridyl, but not by the mitochondrion-selective chelator RPA. Our data strongly suggest that frataxin deficiency does not affect the mitochondrial labile iron pool or other parameters of cellular iron metabolism and suggest a decreased antioxidative defense against extramitochondrial iron-derived radicals in patient cells. These results challenge current concepts favoring the use of mitochondrion-specific iron chelators and antioxidants to treat FRDA. Friedreich's ataxia (FRDA) 1The abbreviations used are: FRDA, Friedreich′s ataxia; RPA, rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester; BSA, bovine serum albumin; TMRM, tetramethylrhodamine methyl ester; 2,2′-DPD, 2,2′-dipyridyl; BME, basal medium Eagle; DTPA, diethylenetriaminepentaacetic acid; Tf, transferrin; Apo-Tf, apo-transferrin; PIH, pyridoxal isonicotinoyl hydrazone; HBSS, Hanks′ balanced salt solution; TfR, transferrin receptor; IRP, iron regulatory protein; IRE, iron responsive element; SOD, superoxide dismutase. is the most common inherited ataxia, affecting one in 50,000 people (1Tan G. Chen L.-S. Lonnerdal B. Gellera C. Taroni F.A. Cortopassi G.A. Hum. Mol. Gen. 2001; 19: 2099-2107Crossref Scopus (73) Google Scholar). Clinically, Friedreich's ataxia is characterized by multiple symptoms including progressive gait and limb ataxia, dysarthria, diabetes mellitus, and hypertrophic cardiomyopathy leading to premature death (2Mateo I. Llorca J. Volpini V. Corral J. Berciano J. Combarros O. Acta Neurol. Scand. 2004; 109: 75-78Crossref PubMed Scopus (32) Google Scholar). There is currently no effective treatment for FRDA. FRDA is caused by a GAA trinucleotide expansion in the frataxin gene located on chromosome locus 9q13, resulting in a reduced expression of frataxin, a small mitochondrial protein (3Harding A.E. Brain. 1981; 104: 589-620Crossref PubMed Google Scholar, 4Campuzano V. Montermini L. Molto M.D. Pianese M. Cossee M. Cavalcanti F. Monros E. Rodius F. Duclos F. Monticeli A. Science. 1996; 271: 1423-1427Crossref PubMed Google Scholar, 5Campuzano V. Montermini L. Lutz Y. Cova L. Hindelang C. Jiralerspong S. Trottier Y. Kish S.J. Faucheux B. Trouillas P. Authier F.J. Durr A. Mandel J.L. Vescovi A. Pandolfo M. Koenig M. Hum. Mol. Genet. 1997; 6: 1771-1780Crossref PubMed Scopus (617) Google Scholar). Because of the mitochondrial localization of frataxin, the neuro- and cardio-degenerations observed in FRDA are thought to be the result of a mitochondrial defect (1Tan G. Chen L.-S. Lonnerdal B. Gellera C. Taroni F.A. Cortopassi G.A. Hum. Mol. Gen. 2001; 19: 2099-2107Crossref Scopus (73) Google Scholar). The importance of frataxin for viability is underscored by the recent finding that a knockout of this gene leads to early embryonic lethality in mice (6Cossée M. Puccio H. Gansmuller A. Koutnikova H. Dierich A. LeMeur M. Fischbeck K. Dolle P. Koenig M. Hum. Mol. Genet. 2000; 9: 1219-1226Crossref PubMed Google Scholar). However, despite a large number of studies that have focused on the function of frataxin in bacteria, yeast, mice, and human cells, its role in the pathogenesis of FRDA is still largely unknown and discussed controversially (7Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar, 8Foury F. FEBS Lett. 1999; 456: 281-284Crossref PubMed Scopus (143) Google Scholar, 9Adamec J. Rusnak F. Owen W.G. Naylor S. Benson L.M. Gacy A.M. Isaya G. Am. J. Hum. Genet. 2000; 67: 549-562Abstract Full Text Full Text PDF PubMed Scopus (230) Google Scholar, 10Voncken M. Ioannou P. Delatycki M.B. Neurogenetics. 2004; 5: 1-8Crossref PubMed Scopus (60) Google Scholar). At least five hypotheses for the primary mitochondrial function of frataxin have been proposed: iron transport (7Babcock M. de Silva D. Oaks R. Davis-Kaplan S. Jiralerspong S. Montermini L. Pandolfo M. Kaplan J. Science. 1997; 276: 1709-1712Crossref PubMed Scopus (828) Google Scholar), iron/sulfur (Fe/S) cluster biosynthesis (8Foury F. FEBS Lett. 1999; 456: 281-284Crossref PubMed Scopus (143) Google Scholar, 11Ro ̈tig A. Sidi D. Munnich A. Rustin P. Trends Mol. Med. 2002; 8: 221-224Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar, 12Bulteau A.-L. O'Neill H.A. Kennedy M.C. Ikeda-Saito M. Isaya G. Szweda L.I. Science. 2004; 305: 242-245Crossref PubMed Scopus (319) Google Scholar), iron storage (1Tan G. Chen L.-S. Lonnerdal B. Gellera C. Taroni F.A. Cortopassi G.A. Hum. Mol. Gen. 2001; 19: 2099-2107Crossref Scopus (73) Google Scholar, 13Gakh O. Adamec J. Gacy A.M. Twesten R.D. Owen W.G. Isaya G. Biochemistry. 2002; 41: 6798-6804Crossref PubMed Scopus (114) Google Scholar), stimulator of oxidative phosphorylation (14Ristow M. Pfister M.F. Yee A.J. Schubert M. Michael L. Zhang C.Y. Ueki K. Michael II, M.D. Lowell B.B. Kahn C.R. Proc. Natl. Acad. Sci. U. S. A. 2000; 97: 12239-12243Crossref PubMed Scopus (194) Google Scholar), and anti-oxidant function (1Tan G. Chen L.-S. Lonnerdal B. Gellera C. Taroni F.A. Cortopassi G.A. Hum. Mol. Gen. 2001; 19: 2099-2107Crossref Scopus (73) Google Scholar). In accordance with the often proposed antioxidant function of frataxin, antioxidant enzyme levels have been found to be significantly different in blood from FRDA patients compared with controls (15Tozzi G. Nuccetelli M. Lo Bello M. Bernardini S. Bellincampi L. Ballerini S. Gaeta L.M. Casali C. Pastore A. Federici G. Bertini E. Piemonte F. Arch. Dis. Child. 2002; 86: 376-379Crossref PubMed Scopus (51) Google Scholar), frataxin has been reported to play a critical role in repair of mitochondrial Fe/S clusters in mitochondrial aconitase (12Bulteau A.-L. O'Neill H.A. Kennedy M.C. Ikeda-Saito M. Isaya G. Szweda L.I. Science. 2004; 305: 242-245Crossref PubMed Scopus (319) Google Scholar), and it has been shown that superoxide dismutase (SOD) genes are not induced by oxidative stress in FRDA fibroblasts, in contrast to control cells (10Voncken M. Ioannou P. Delatycki M.B. Neurogenetics. 2004; 5: 1-8Crossref PubMed Scopus (60) Google Scholar, 16Jiralerspong S. Ge B. Hudson T.J. Pandolfo M. FEBS Lett. 2001; 509: 101-105Crossref PubMed Scopus (68) Google Scholar, 17Chantrel-Groussard K. Geromel V. Puccio H. Koenig M. Munnich A. Ro ̈tig A. Rustin P. Hum. Mol. Genet. 2001; 10: 2061-2067Crossref PubMed Google Scholar). The prevailing hypothesis underlying the pathogenesis of FRDA, however, proposes that frataxin is somehow involved in the regulation of mitochondrial iron homeostasis and that impaired intramitochondrial iron metabolism results in iron overload and oxidative stress (10Voncken M. Ioannou P. Delatycki M.B. Neurogenetics. 2004; 5: 1-8Crossref PubMed Scopus (60) Google Scholar, 18Patel P.I. Isaya G. Am. J. Hum. Genet. 2001; 69: 241515-241524Google Scholar, 19Puccio H. Koenig M. Hum. Mol. Genet. 2000; 9: 887-892Crossref PubMed Google Scholar). An accumulation of iron detected as iron deposits using Perl's staining has been consistently observed on autopsy in heart muscle of FRDA patients (20Lamarche J.B. Shapcott D. Cote M. Lemieux B. Lechtenberg R. Handbook of Cerebellar Diseases. Marcel Dekker, New York1993: 453-456Google Scholar) and magnetic resonance imaging data indicate that iron also accumulates in the dentate nucleus (21Waldvogel D. van Gelderen P. Hallett M. Ann. Neurol. 1999; 46: 123-125Crossref PubMed Scopus (191) Google Scholar). A small but significant intramitochondrial accumulation of total iron has also been reported in one patient study (22Delatycki M.B. Camakaris J. Brooks H. Evans-Whipp T. Thorburn D.R. Williamson R. Forrest S.M. Ann. Neurol. 1999; 45: 673-675Crossref PubMed Scopus (156) Google Scholar), while no significant increase in total mitochondrial iron under physiological cell culture conditions was found in others (1Tan G. Chen L.-S. Lonnerdal B. Gellera C. Taroni F.A. Cortopassi G.A. Hum. Mol. Gen. 2001; 19: 2099-2107Crossref Scopus (73) Google Scholar, 19Puccio H. Koenig M. Hum. Mol. Genet. 2000; 9: 887-892Crossref PubMed Google Scholar, 23Wong A. Yang J. Danielson S. Gellera C. Taroni F. Cortopassi G. Antiox. Redox Signal. 2000; 2: 461-465Crossref PubMed Google Scholar). Intramitochondrial iron accumulation has been postulated to be the primary pathogenetic event initiating the production of hydroxyl radicals by Fenton chemistry, leading to inactivation of Fe/S proteins, lipid peroxidation and damage to nucleic acids and proteins, finally resulting in cell death (10Voncken M. Ioannou P. Delatycki M.B. Neurogenetics. 2004; 5: 1-8Crossref PubMed Scopus (60) Google Scholar, 24Bradley J.L. Blake J.C. Chamberlain S. Thomas P.K. Cooper J.M. Schapira A.H.V. Hum. Mol. Genet. 2000; 9: 275-282Crossref PubMed Google Scholar). However, in a conditional knock-out mouse model for FRDA, accumulation of intramitochondrial total iron has been reported to follow a decrease in the Fe/S cluster-containing subunits of the mitochondrial electron transport chain (complexes I-III) and of the enzyme aconitase (25Puccio H. Simon D. Cossee M. Criqui-Filipe P. Tiziano F. Melki J. Hindelang C. Matyas R. Rustin P. Koenig M. Nat. Genet. 2001; 27: 181-186Crossref PubMed Scopus (603) Google Scholar), and thus was suggested to be a late event in the pathogenetic process. The form of iron generally regarded to be responsible for the generation of highly reactive oxygen species mediating oxidative stress is termed "labile" or "chelatable iron," because of its accessibility to iron chelators (26Petrat F. de Groot H. Sustmann R. Rauen U. Biol. Chem. 2002; 383: 489-502Crossref PubMed Scopus (191) Google Scholar, 27Kakhlon O. Cabantchik Z.I. Free Radic. Biol. Med. 2002; 33: 1037-1046Crossref PubMed Scopus (623) Google Scholar, 28Halliwell B. Gutteridge J.M.C. Free Radicals in Biology and Medicine. Oxford University Press, Oxford1999: 132-133Google Scholar). However, although iron is obviously involved in the pathogenesis of FRDA, the connection between frataxin deficiency and the size of the mitochondrial labile (chelatable) iron pool and its role in the pathogenesis of FRDA have not yet been characterized because of the lack of a suitable methodological approach. Therefore, using the novel mitochondrion-specific iron indicator/chelator rhodamine B-[(1,10-phenanthrolin-5-yl)aminocarbonyl]benzylester (RPA, Refs. 29Petrat F. Weisheit D. Lensen M. de Groot H. Sustmann R. Rauen U. Biochem. J. 2002; 362: 137-147Crossref PubMed Scopus (119) Google Scholar and 30Rauen U. Kerkweg U. Weisheit D. Petrat F. Sustmann R. de Groot H. Free Radic. Biol. Med. 2003; 35: 1664-1678Crossref PubMed Scopus (64) Google Scholar), we have determined here for the first time the concentration of mitochondrial chelatable iron in FRDA cells, i.e. in FRDA patient lymphoblast and fibroblast cell lines in comparison with age- and sex-matched control cell lines. In addition, we studied the influence of decreased frataxin levels on various other parameters of iron metabolism as well as the contribution of mitochondrial chelatable iron to cell death under H2O2-induced oxidative stress in FRDA patient cells. Materials—The mitochondrion-selective fluorescent iron indicator RPA was a generous gift from Prof. R. Sustmann (Institut für Organische Chemie, Universität Duisburg-Essen, Germany); RPA is available from Squarix Biotechnology GmbH (Marl, Germany). RPMI 1640 medium was from Invitrogen, Life Technologies (Karlsruhe, Germany), bovine serum albumin (BSA) from Serva (Heidelberg, Germany), and 1× protease inhibitor mixture and RNaseT1 from Roche Applied Science (Basel, Switzerland). Dimethyl sulfoxide and Triton X-100 were from Merck (Darmstadt, Germany). SDS and a protein assay reagent were obtained from Bio-Rad. Fetal calf serum, ferrous ammonium sulfate, poly-l-lysine, tetramethylrhodamine methyl ester (TMRM), 2,2′-dipyridyl (2,2′-DPD), propidium iodide, basal medium Eagle (BME), chelex (chelating resin; iminodiacetic acid), hydrogen peroxide, diethylenetriaminepentaacetic acid (DTPA), apo-transferrin (Apo-Tf), ferric ammonium citrate, HEPES, dithiothreitol, and horseradish peroxidase-conjugated goat anti-mouse IgG came from Sigma. The fluorescent dye rhodamine 123 was purchased from Molecular Probes Europe BV (Leiden, Netherlands), the ECL Western blotting system from Amersham Biosciences (Freiburg, Germany), and the mouse anti-frataxin monoclonal antibody 2FRA-1G2 from Chemicon International Inc. (Temecula, CA). Radioactive iron (55FeCl3) and [α-32P]cytidinetriphosphate were obtained from PerkinElmer Life Sciences and Readysafe from Beckman. Pyridoxal isonicotinoyl hydrazone (PIH) was a generous gift from Prof. Dr. P. Ponka (Lady Davis Institute for Medical Research, Montreal, Canada). PIH (100.0 mm) was dissolved in 0.1 n NaOH, then diluted with Hanks' balanced salt solution (HBSS: 137.0 mm NaCl, 5.4 mm KCl, 1.0 mm CaCl2, 0.5 mm MgCl2, 0.4 mm KH2PO4, 0.4 mm MgSO4, 0.3 mm Na2HPO4, 25.0 mm HEPES, pH 7.4) and adjusted to pH 7.35 (37 °C). Falcon cell culture flasks and Falcon 6-well cell culture plates were obtained from BD Biosciences, glass coverslips were from Assistent (Sondheim/Röhn, Germany), and gas mixtures from Messer Griesheim (Krefeld, Germany). Cell Culture—Lymphoblast and skin fibroblast cell lines from Friedreich's ataxia patients and age- and sex-matched controls were established after informed consent at the Murdoch Childrens Research Institute, Royal Children's Hospital, Melbourne, Australia (Table I). All studies were approved by the Royal Children's Hospital Ethics in Human Research Committee. Fibroblast cell lines were cultured on 6.2 cm2 glass coverslips in 6-well cell culture plates or in 12.5 cm2 culture flasks in BME supplemented with 10% fetal calf serum, l-glutamine (2.0 mm), NaHCO3 (11.6 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and HEPES (20 mm; pH 7.35, 37 °C). Lymphoblast cell lines were cultured in suspension in 25 cm2 culture flasks (kept in an upright position) in RPMI 1640 medium supplemented with 20% fetal calf serum, l-glutamine (1.6 mm), NaHCO3 (7.5 mm), sodium pyruvate (0.8 mm), uridine (0.04 mm), penicillin (100 units/ml), streptomycin (100 μg/ml), and HEPES (16 mm; pH 7.35, 37 °C). All cell lines were cultured in a humidified atmosphere of 5% CO2, 95% air.Table IHuman lymphoblast and fibroblast cell lines studiedCell typeCell lineFRDA statusSexAge (years)GAA expansion size (triplets of smaller/larger alleles)LymphoblastsKS10TPatientFemale37650, 1290KS14TControlFemale37KS30TPatientFemale20810, 1070KS33TControlFemale21KS100TPatientMale41610, 990KS43TControlMale40KS160TPatientMale43620, 1190KS103TControlMale41Fibroblasts970474PatientFemale30NDaND, not determined.S103ControlFemale24970476PatientFemale40700, 1100920163ControlFemale35FR-1aPatientMale35670, 870S90ControlMale15FR-3aPatientMale40560, 670920162ControlMale32a ND, not determined. Open table in a new tab Determination of Mitochondrial Chelatable Iron—Mitochondrial chelatable iron of transformed lymphoblast and fibroblast cell lines from Friedreich's ataxia patients and age- and sex-matched controls was determined using the mitochondrion-selective fluorescent iron indicator RPA, the fluorescence of which is quenched by chelatable iron (26Petrat F. de Groot H. Sustmann R. Rauen U. Biol. Chem. 2002; 383: 489-502Crossref PubMed Scopus (191) Google Scholar, 29Petrat F. Weisheit D. Lensen M. de Groot H. Sustmann R. Rauen U. Biochem. J. 2002; 362: 137-147Crossref PubMed Scopus (119) Google Scholar, 30Rauen U. Kerkweg U. Weisheit D. Petrat F. Sustmann R. de Groot H. Free Radic. Biol. Med. 2003; 35: 1664-1678Crossref PubMed Scopus (64) Google Scholar, 31Rauen U. Petrat F. Sustmann R. de Groot H. J. Hepatol. 2004; 40: 607-615Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar). Human fibroblasts (1.0 × 105 cells), cultured on glass coverslips until confluency, were loaded with RPA (0.2 μm) in HBSS for 20 min at 37 °C, followed by an additional 15 min of incubation in dye-free buffer. Human lymphoblasts were washed once with HBSS by centrifugation (250 × g for 5 min at 37 °C) and attached (5 × 106 cells/ml) to poly-l-lysine-coated (5 μg/cm2) glass coverslips 15 min before loading the cells with RPA. Adherent lymphoblasts were incubated with RPA as described for the fibroblast cell lines. For measurements, cells were incubated in HBSS (37 °C), and measurements were performed by laser scanning microscopy (λexc. = 543 nm and λem. ≥ 560 nm (29Petrat F. Weisheit D. Lensen M. de Groot H. Sustmann R. Rauen U. Biochem. J. 2002; 362: 137-147Crossref PubMed Scopus (119) Google Scholar)). After establishing the mitochondrial baseline fluorescence, the mitochondrial chelatable iron was removed from the indicator by the addition of a large excess of the membrane-permeable iron chelator PIH (2 mm) leading to a "dequenching" of the RPA fluorescence. The increase in fluorescence was taken as a measure of the concentration of the intramitochondrial chelatable iron, which was quantified using an ex situ calibration. In other experiments, cells were incubated simultaneously with RPA and rhodamine 123 (0.2 μm) in order to prove selective mitochondrial staining. In experiments with double-stained mitochondria, red fluorescence of RPA (λexc. = 543 nm; λem. ≥ 585 nm) and green fluorescence of rhodamine 123 (λexc. = 488 nm; λem. = 505–530 nm) were optically isolated in successive scans. At the end of the experiments, the uptake of the vital dye propidium iodide (5 μg/ml) was routinely determined in order to assess loss in cell viability. The red fluorescence of propidium iodide excited at 543 nm was collected through a 560 nm long-pass filter. Determination of 55Fe-Tf-uptake and Functional Transferrin Receptor Density—The uptake of transferrin-bound iron by FRDA patient and control cells was studied using 55Fe-loaded human apo-Tf. Human apo-Tf was loaded with 55FeCl3 according to Bates and Schlabach (32Bates G.W. Schlabach M.R. J. Biol. Chem. 1975; 250: 2177-2181Abstract Full Text PDF PubMed Google Scholar). Fibroblasts and lymphoblasts were washed twice with their respective medium containing 50 μm DTPA to remove extracellular iron and then washed once with medium alone. For the 55Fe-Tf-uptake, the cells (5 × 106/ml) were incubated for 30 min in serum-free medium (37 °C) supplemented with 1 mg/ml BSA, 20 mm HEPES, and 55Fe-Tf (100–800 nm iron). Transferrin receptor (TfR) saturation kinetics was assessed by incubating the cells with 55Fe-Tf (0–1600 nm iron) for 0–45 min under the same conditions; samples were taken every 15 min. The cellular uptake of 55Fe-Tf was terminated by separating the cells from the supernatant. Lymphoblast suspensions were centrifuged (3,000 × g for 3 min at 4 °C) and the pellet was purified by further centrifugation through an oil layer consisting of 80% w/w dibutylphthalate and 20% w/w dioctylphthalate. Fibroblasts were washed with serum-free medium containing 50 μm DTPA to remove extracellular iron and then washed once with BME alone. Cells were lysed overnight in 500 μlof0.5 m KOH with 1% Triton X-100, and the lysates were neutralized with 250 μl of 1 m HCl. Intracellular radioactivity was counted in a Packard liquid scintillation counter (Vienna, Austria) subsequent to the addition of Readysafe. Kinetic analyses were calculated with GraphPad Prism (GraphPad Software, San Diego, CA). Assessment of H2O2 Cytotoxicity—FRDA fibroblasts and control cells were cultured in 12.5 cm2 cell culture flasks until confluency. Before starting the experiments, cells were washed twice with HBSS (37 °C) and then covered with Krebs-Henseleit buffer (115 mm NaCl, 25 mm NaHCO3, 5.9 mm KCl, 1.2 mm NaH2PO4, 1.2 mm MgCl2, 2.5 mm CaCl2, 1.2 mm Na2SO4, 20 mm HEPES, pH 7.4), supplemented with 10 mm glucose as previously reported for murine L929 fibroblasts (33Lomonosova E.E. Kirsch M. de Groot H. Free Radic. Biol. Med. 1998; 25: 493-503Crossref PubMed Scopus (35) Google Scholar). Afterward, H2O2 was added as a bolus of 500 μm from a concentrated stock solution, and cells were incubated at 37 °C in an atmosphere of 5% CO2/95% air. In some experiments, the transition metal chelators 2,2′-DPD (200 μm) or 1,10-phenanthroline (100 μm) were added to the Krebs-Henseleit buffer prior to the addition of H2O2 or the cells were preincubated with deferoxamine (10 mm for 30 min in BME, followed by washing with HBSS). Other cultures were preincubated with RPA (0.2 μm, in HBSS, 20 min at 37 °C, followed by an additional 15 min of incubation in dye-free buffer, see above) prior to the start of the experiments. Solvent controls were included. After various periods of incubation, loss in cell viability was assessed by the release of lactate dehydrogenase (LDH) activity using a standard assay. Western Blot Analysis—The endogenous frataxin of cultured human fibroblasts of Friedreich's ataxia patients and healthy control individuals was detected on Western blots using the mouse anti-frataxin monoclonal antibody 2FRA-1G2. Whole cell protein extracts were prepared from 75 cm2 cell culture flasks of subconfluent cells. Cells were lysed in 200 μl of extraction buffer (300 mm NaCl, 10 mm Tris, pH 7.9, 1 mm EDTA, 0.1% Nonidet P-40, 1× protease inhibitor mixture) for 20 min on ice and centrifuged (3,600 × g at 4 °C for 5 min). The supernatant was used as whole cell extract, and the protein concentration was determined using a protein assay reagent. 100 μg of protein per lane were loaded onto a 12.5% SDS-polyacrylamide gel, and after electrophoresis the gel was blotted onto nitrocellulose membranes, which were blocked with 5% nonfat milk overnight at 4 °C. Incubation with the primary monoclonal antibodies against frataxin (diluted 1:2,500 in 5% BSA) was performed for 1 h at room temperature. Horseradish peroxidase-conjugated goat anti-mouse IgG was used as a secondary antibody (1:10,000 dilution, incubation for 1 h). An ECL Western blotting system was used for detection of the immunoreactive proteins according to the manufacturer's instructions. IRP/IRE Bandshift Assay—The activity of iron regulatory proteins (IRP)/iron responsive elements (IRE) in FRDA fibroblasts and control cells was determined according to the method described by Müllner et al. (34Mu ̈llner E.W. Neupert B. Kuhn L.C. Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (403) Google Scholar). Cells were washed twice with BME containing 50 μm DTPA to remove extracellular iron and then once with the medium alone. To study basal and iron-induced changes in IRP/IRE activity, cells were then incubated in the absence or presence of 150 μm ferric ammonium citrate for 6 h in serum-free medium (37 °C) supplemented with 1 mg/ml BSA and 20 mm HEPES. Afterward, the fibroblasts were lysed in 0.2% Nonidet P-40 buffer (40 mm KCl, 5% glycerol, 1 mm dithiothreitol, 0.2% Nonidet P-40, 10 mm HEPES, pH 7.6). Radioactive labeled IRE probes were prepared by in vitro transcription of pSPT-Fer (IRE of human ferritin H-chain mRNA (34Mu ̈llner E.W. Neupert B. Kuhn L.C. Cell. 1989; 58: 373-382Abstract Full Text PDF PubMed Scopus (403) Google Scholar)) in the presence of [α-32P]cytidinetriphosphate. IRE/IRP binding reactions were carried out with 2 μg of protein and 640,000 cpm of 32P-labeled IRE probe by incubation for 20 min at room temperature. Thereafter, samples were treated with 20 units of RNase T1 and 5 μg/μl heparin for 10 min each. RNA-protein complexes were separated in 6% non-denaturing polyacrylamide gels and fixed onto diethylaminoethyl ion exchange paper. Signals corresponding to IRP-IRE complexes were quantified by phosphorimaging (ImageQuant Software, Amersham Biosciences). The total amount of IRP1 was assessed by in vitro reduction with 3% 2-mercaptoethanol prior to the binding reaction. Statistics—All experiments with human lymphoblasts and fibroblasts were performed in duplicate with cell lines from different patients and different age- and sex-matched controls. Cellular microfluorographs and blots shown in the figures are representative of all the corresponding experiments carried out. Other results are expressed as means ± S.D. Data obtained from two groups were compared by means of Student's t test (matched values, two-tailed, paired). A p value of < 0.05 was considered significant. GAA Expansion Size and Frataxin Content of Human Fibroblasts and Lymphoblasts Derived from Friedreich's Ataxia Patients—The size of the GAA expansions as determined by polymerase chain reaction on genomic DNA was found to range from about 600–1300 repeats in the lymphoblast and fibroblast cultures of all patients (Table I), thus confirming large GAA alleles on both chromosomes. To verify that the frataxin level in the FRDA patient-derived cell lines was actually decreased, Western blot analysis was carried out. Using the 2FRA-1G2 human antibody we detected a ∼18 kDa protein, i.e. a protein with a molecular mass consistent with the one of human frataxin, in the fibroblasts of the healthy control individuals, whereas the residual frataxin levels of fibroblasts of the Friedreich's ataxia patients were barely detectable (Fig. 1). This result confirmed that the cells derived from Friedreich's ataxia patients and age- and sex-matched controls differed profoundly in their frataxin content, thus excluding the possibility of an artifact arising during the establishment and maintenance of the cell lines. Mitochondrial Chelatable Iron in Cultured Human FRDA and Control Fibroblasts and Lymphoblasts—The nature of the iron that has been reported to accumulate within the mitochondria of patients with Friedreich's ataxia is yet unknown. All of the methods previously used to study changes in the intramitochondrial iron homeostasis primarily assess the total mitochondrial iron content but are not suitable to selectively determine the small fraction of mitochondrial iron that is regarded to be responsible for the initiation of iron-mediated oxidative injury, i.e. the labile (chelatable) iron (26Petrat F. de Groot H. Sustmann R. Rauen U. Biol. Chem. 2002; 383: 489-502Crossref PubMed Scopus (191) Google Scholar, 27Kakhlon O. Cabantchik Z.I. Free Radic. Biol. Med. 2002; 33: 1037-1046Crossref PubMed Scopus (623) Google Scholar, 29Petrat F. Weisheit D. Lensen M. de Groot H. Sustmann R. Rauen U. Biochem. J. 2002; 362: 137-147Crossref PubMed Scopus (119) Google Scholar, 35Petrat F. de Groot H. Rauen U. Biochem. J. 2001; 356: 61-69Crossref PubMed Scopus (186) Google Scholar). 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