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Dual Role of the Mitochondrial Protein Frataxin in Astrocytic Tumors

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Dual Role of the Mitochondrial Protein Frataxin in Astrocytic Tumors Laboratory Investigation (2011) 91, 1766–1776 & 2011 USCAP, Inc All rights reserved 0023-6837/11 $32.00 Dual role of the mitochondrial protein frataxin in astrocytic tumors Elmar Kirches1, Nadine Andrae1, Aline Hoefer2, Barbara Kehler1,3, Kim Zarse4, Martin Leverkus3, Gerburg Keilhoff5, Peter Schonfeld5, Thomas Schneider6, Annette Wilisch-Neumann1 and Christian Mawrin1 The mitochondrial protein frataxin (FXN) is known to be involved in mitochondrial iron homeostasis and iron–sulfur cluster biogenesis. It is discussed to modulate function of the electron transport chain and production of reactive oxygen species (ROS). FXN loss in neurons and heart muscle cells causes an autosomal-dominant mitochondrial disorder, Friedreich’s ataxia. Recently, tumor induction after targeted FXN deletion in liver and reversal of the tumorigenic phenotype of colonic carcinoma cells following FXN overexpression were described in the literature, suggesting a tumor suppressor function. We hypothesized that a partial reversal of the malignant phenotype of glioma cells should occur after FXN transfection, if the mitochondrial protein has tumor suppressor functions in these brain tumors. In astrocytic brain tumors and tumor cell lines, we observed reduced FXN levels compared with non-neoplastic astrocytes. Mitochondrial content (citrate synthase activity) was not significantly altered in U87MG glioblastoma cells stably overexpressing FXN (U87-FXN). Surprisingly, U87-FXN cells exhibited increased cytoplasmic ROS levels, although mitochondrial ROS release was attenuated by FXN, as expected. Higher cytoplasmic ROS levels corresponded to reduced activities of glutathione peroxidase and catalase, and lower glutathione content. The defect of antioxidative capacity resulted in increased susceptibility of U87-FXN cells against oxidative stress induced by H2O2 or buthionine sulfoximine. These characteristics may explain a higher sensitivity toward staurosporine and alkylating drugs, at least in part. On the other hand, U87-FXN cells exhibited enhanced growth rates in vitro under growth factor-restricted and hypoxic conditions and in vivo using tumor xenografts in nude mice. These data contrast to a general tumor suppressor function of FXN but suggest a dual, pro-proliferative but chemosensitizing role in astrocytic tumors. Laboratory Investigation (2011) 91, 1766–1776; doi:10.1038/labinvest.2011.130; published online 22 August 2011 KEYWORDS: chemotherapy; frataxin; glioma; oxidative stress A GAA repeat expansion in the frataxin gene causes the lethal biogenesis,6 the central steps of which take place in the disorder Friedreich’s ataxia (FRDA), which is characterized mitochondrial matrix. FXN oligomers exhibit large capacity by degeneration of spinocerebellar tracts, dorsal columns and for binding ferrous iron. After binding of FXN to the ISC- pyramidal tracts, as well as heart failure. The mutation scaffold protein, iron may be directly handed over to the abolishes gene transcription, leading to a loss of the metal-free ISCs, which are then transferred to their final mitochondrial protein frataxin (FXN), mitochondrial iron targets.7 ISC proteins include complexes of the electron accumulation and oxidative stress.1,2 Knockout of the FXN transport chain (ETC) or ferrochelatase, an enzyme of heme homolog Yfh1p in yeasts impairs respiration and causes biogenesis.8 FXN is thus essential for the function of ISC- mitochondrial DNA depletion and iron accumulation.3,4 and heme-containing ETC complexes and of cytochrome c. Defects of iron–sulfur proteins in myocardial biopsies of In tumor cells, FXN has been discussed as a modulator of FRDA patients confirmed a defective iron–sulfur cluster energy metabolism, with an impact on growth characteristics. (ISC) biogenesis in the Yfh1p knockout model.5 According to FXN-transformed colon cancer cell lines exhibited enhanced the current view, mammalian FXN is directly involved in ISC respiration and were characterized by decreased growth rates 1Department of Neuropathology, Otto-von-Guericke University, Magdeburg, Germany; 2Department of Neuropathology, Friedrich-Schiller University, Jena, Germany; 3Department of Experimental Dermatology, Otto-von-Guericke University, Magdeburg, Germany; 4Department of Human Nutrition, Friedrich-Schiller University, Jena, Germany; 5Department of Biochemistry and Cell Biology, Otto-von-Guericke University, Magdeburg, Germany and 6Department of Neurosurgery, Otto-von-Guericke University, Magdeburg, Germany Correspondence: Dr C Mawrin, MD, Department of Neuropathology, Otto-von-Guericke-University, Leipziger Strasse 44, D-39120 Magdeburg, Germany. E-mail: [email protected] Received 27 January 2011; revised 04 July 2011; accepted 13 July 2011 1766 Laboratory Investigation | Volume 91 December 2011 | www.laboratoryinvestigation.org Frataxin in astrocytomas E Kirches et al in soft agar assays and nude mice.9 These results were ATCC. Three of these markers, amelogenin (X), vWA (alleles interpreted in terms of cancer growth inhibition by 15, 17) and THO1 (allele 9.3) are published for the parental antioxidative properties of FXN, leading to an enhancement line U87MG in the ATCC database (http://www.ATCC.org). of oxidative phosphorylation (OXPHOS). The reciprocal OLN-93 was originally derived from a primary rat brain glial experiment was a hepatocyte-specific FXN knockout in mice. culture and resembles primary oligodendrocytes. It was a The induction of multiple hepatic tumors with decreased kind gift of Professor Richter-Landsberg (Department mitochondrial metabolism and iron–sulfur proteins sug- of Biology, University of Oldenburg, Germany), who first gested a tumor suppressor function for FXN.10 described the line. Malignant astrocytic tumors are among the most frequent intracranial neoplasms. Despite recent advances of chemo- Human Tumor Tissues therapy, the improvement of median overall survival of The frozen samples were taken from the files of the patients with glioblastoma (GBM) remains a major chal- Department of Neuropathology of the Otto-von-Guericke 11 lenge. In this report, we investigated the role of FXN in University. The samples had been obtained during routine astrocytic tumors and GBM cell lines. We hypothesized that neurosurgical excision of samples for diagnostic purposes. the malignant biological behavior of astrocytic tumor cells, Diagnosis had been performed by an experienced neuro- especially regarding tumor growth, should be attenuated by pathologist (CM). FXN transfection in vitro and in vivo, if the protein has tumor suppressor properties in these cells. If these biological Mixed Rat Brain Cell Cultures effects were connected with the suggested antioxidative and Neuronal/glial mixed cultures were prepared from cerebellum OXPHOS-protecting properties of FXN, transfected cells of 4-day-old rats in DMEM with 10% FCS and 1% (v/v) should exert lower reactive oxygen species (ROS) generation penicillin/streptomycin. After mechanical dispersion, ali- and higher aconitase activity. In addition, it is likely that quots of the suspension were plated in poly-D-lysine coated the transfected cells are less sensitive to externally applied dishes at a final density of 2 Â 106 cells per 8 cm2. After 48 h, oxidative stress. non-adherent cells were removed by medium change. After 10 days, the cells were used for experiments. For serum/glu- MATERIALS AND METHODS cose deprivation (SGD), growth medium was replaced by Vector Construction serum- and glucose-free DMEM for 24 h. pCl-neo-FXN was generated by cloning a hemagglutin- tagged human frataxin cDNA into pCl-neo (Promega, Mannheim, Germany). Qualitative and Real-Time PCR RNA was isolated using the RNeasy-mini-kits from Qiagen Cell Culture (Hilden, Germany). FXN-PCR from cDNA was performed The GBM cell lines U87MG, U118MG, U138MG and LN405 with the primers TAAGCGTTATGACTGGACTGG and GAA b were purchased from the American Type Culture Collection TAGGCCAAGGAAGACAAG, -actin (ACC ACC CCA GCC (ATCC) or the German Collection of Microorganisms and ATG TAC G and ATG TCA CGC ACG ATT TCC C) serving Cell Cultures, respectively. Transformed cell lines U87-FXN as a reference. For SYBR-green-based Real-Time PCR on an and U87-Neo were generated by lipofectamine-2000 trans- ABI-7000-SDS (Applied Biosystems, CA), the same primers 1 fection of the plasmids into U87MG cells and culturing were used with identical annealing temperature (57 C). under 0.2 mg/ml G418. This concentration had been determined as the minimal concentration, which kills Western Blotting parental cells within 2 weeks. Developing colonies of resistant Cells were washed twice with ice-cold PBS and harvested by cells were selectively transferred to new flask and maintained scraping in RIPA buffer (10 mM Tris-HCl, pH 7.4; 150 mM under G418. The cell lines used in the present study thus NaCl; 50 mM NaF; 1 mM EDTA; 1% Triton X-100; 0.1% represent cell clones. Human cortical astrocytes were SDS; 0.5% deoxycholate) with DTT, Na-vanadate and pro- purchased from ScienCell Research Laboratories (Carlsbad, tease inhibitors. In all, 40 mg protein was separated by SDS- CA) and embryonal rat astrocytes prepared in our labora- PAGE and transferred onto nitrocellulose membranes. After tory.12 All cells were cultured in DMEM with 10% FCS. The blocking, membranes were incubated with primary anti- identity of the cell lines was analyzed using the AmpFSTR kit bodies in TBST overnight at 4 1C, followed by chemilumi- and the software GeneMapper ID v3.2 (Applied Biosystems), nescence (Millipore, Schwalbach,
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