TY - THES A1 - Thierbach, René T1 - Identifikation des mitochondrialen Proteins Frataxin als stoffwechselmodulierenden Tumorsuppressor N2 - Die Krebsentstehung wurde vor rund 80 Jahren auf veränderten zellulären Energiestoffwechsel zurückgeführt. Diese Hypothese konnte bisher weder experimentell bewiesen noch widerlegt werden. Durch den Einsatz zweier Modellsysteme mit unterschiedlicher Expression des mitochondrialen Proteins Frataxin konnte in der vorliegenden Arbeit gezeigt werden, dass der mitochondriale Energiestoffwechsel einen Einfluss auf die Tumorentstehung zu besitzen scheint. Eine Reduktion des mitochondrialen Energiestoffwechsels wurde durch die hepatozytenspezifische Ausschaltung des mitochondrialen Proteins Frataxin in Mäusen erreicht. Der durch das Cre-/loxP-Rekombinasesystem erreichte organspezifische Knock-out wurde auf Transkriptions- und Translationsebene nachgewiesen. Anhand verminderter Aconitaseaktivität, geringeren Sauerstoffverbrauches und reduzierten ATP-Gehaltes im Lebergewebe wurde ein signifikant verminderter Energiestoffwechsel dargestellt. Zwar entsprach die Genotypenverteilung in den Versuchsgruppen der erwarteten Mendelschen Verteilung, dennoch war die mittlere Lebenserwartung der Knock-out-Tiere mit ca. 30 Wochen stark reduziert. Bereits in jungem Alter war bei diesen Tieren die Ausbildung von präneoplastischen Herden zu beobachten. Mit proteinbiochemischen Nachweistechniken konnte in Lebergewebe 4-8 Wochen alter Tiere eine verstärkte Aktivierung des Apoptosesignalweges (Cytochrom C im Zytosol, verstärkte Expression von Bax) sowie eine Modulation stressassoziierter Proteine (geringere Phosphorylierungsrate p38-MAPK, vermehrte Expression HSP-25, verminderte Expression HSP-70) aufgezeigt werden. Im inversen Ansatz wurde eine Steigerung des mitochondrialen Energiestoffwechsels durch stabile transgene Frataxinüberexpression in zwei Kolonkarzinomzelllinien erreicht. Diese Steigerung zeigte sich durch erhöhte Aconitaseaktivität, erhöhten Sauerstoffverbrauch, gesteigertes mitochondriales Membranpotenzial und erhöhten ATP-Gehalt in den Zellen. Die frataxinüberexprimierenden Zellen wuchsen signifikant langsamer als Kontrollzellen und zeigten im Soft-Agar-Assay und im Nacktmausmodell ein deutlich geringeres Potenzial zur Ausbildung von Kolonien bzw. Tumoren. Mittels Immunoblot war hier eine vermehrte Phosphorylierung der p38-MAPK festzustellen. Die zusammenfassende Betrachtung beider Modelle zeigt, dass ein reduzierter mitochondrialer Energiestoffwechsel durch Regulation der p38-MAPK und apoptotischer Signalwege ein erhöhtes Krebsrisiko zu verursachen vermag. N2 - Eigthy years ago, it was suggested that impaired energy metabolism might cause cancer. Compelling experimental evidence for this hypothesis is lacking. By use of two different model systems here we show that impaired expression of the mitochondrial protein frataxin leading to impaired mitochondrial energy metabolism appears to be inversely related to tumour growth. To generate mice with reduced mitochondrial energy metabolism the expression of mitochondrial protein frataxin was disrupted in a hepatocyte-specific manner by using the cre/loxP-system. Presence, efficiency and specificity of disruption were shown at transcriptional and translational levels. Decreased activity of aconitase, reduced oxygen consumption and diminished ATP level in the liver revealed diminished energy metabolism. Although knock-out mice were born in the expected Mendelian frequency, they exhibited a significantly decreased life expectancy. Young mice exhibited hepatic preneoplasia. The use of proteinbiochemical techniques revealed activation of apoptotic pathways (cytochrome c in the cytosol, increased expression of bax) and modulation of stress-associated cascades (decreased phosphorylation of p38-MAPK, increased expression of HSP-25 and diminished expression of HSP-70). Inversely, transgenic overexpression of frataxin in colon cancer cell lines lead to increased mitochondrial energy metabolism as demonstrated by elevated activity of aconitase, increased oxygen consumption, elevated mitochondrial membrane potential and increased ATP levels. Frataxin-overexpressing colon cancer cells exhibit a concurrent decrease in replication rate. The colony forming capacity in soft-agar-assay and tumour formation in nude mice were clearly decreased. Immunoblotting revealed elevated phosphorylation of p38-MAPK. Taken together, these models suggest that reduced mitochondrial energy metabolism may promote cancer through regulation of p38-MAPK and apoptotic pathways. T2 - Identifikation des mitochondrialen Proteins Frataxin als stoffwechselmodulierenden Tumorsuppressor KW - Energiestoffwechsel KW - Krebs KW - Frataxin KW - Knock-out KW - Zelllinien KW - Tumor KW - Mitochondrien KW - metabolism KW - cancer KW - frataxin KW - knock-out KW - cell line KW - tumor KW - energy KW - mitochondria Y1 - 2004 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:517-0001943 ER - TY - JOUR A1 - Thierbach, Rene A1 - Schulz, Tim Julius A1 - Voigt, Aanja A1 - Drewes, Gunnar A1 - Isken, F. A1 - Pfeiffer, Andreas F. H. A1 - Ristow, Michael A1 - Steinberg, Pablo T1 - Targeted disruption of frataxin in hepatocytes causes spontaneous neoplasia accompanied by increased ROS formation Y1 - 2004 SN - 0028-1298 ER - TY - JOUR A1 - Thierbach, Renè A1 - Schulz, Tim Julius A1 - Isken, Frank A1 - Voigt, Aanja A1 - Mietzner, Brun A1 - Drewes, Gunnar A1 - von Kleist-Retzow, Jürgen-Christoph A1 - Wiesner, Rudolf J. A1 - Magnuson, Mark A. A1 - Puccio, Helene A1 - Pfeiffer, Andreas F. H. A1 - Steinberg, Pablo A1 - Ristow, Michael T1 - Targeted disruption of hepatic frataxin expression causes impaired mitochondrial function, decreased life span and tumor growth in mice N2 - We have disrupted expression of the mitochondrial Friedreich ataxia protein frataxin specifically in murine hepatocytes to generate mice with impaired mitochondrial function and decreased oxidative phosphorylation. These animals have a reduced life span and develop multiple hepatic tumors. Livers also show increased oxidative stress, impaired respiration and reduced ATP levels paralleled by reduced activity of iron-sulfur cluster (Fe/S) containing proteins (ISP), which all leads to increased hepatocyte turnover by promoting both apoptosis and proliferation. Accordingly, phosphorylation of the stress-inducible p38 MAP kinase was found to be specifically impaired following disruption of frataxin. Taken together, these findings indicate that frataxin may act as a mitochondrial tumor suppressor protein in mammals Y1 - 2005 ER - TY - JOUR A1 - Schulz, Tim Julius A1 - Thierbach, Renè A1 - Voigt, Anja A1 - Drewes, Gunnar A1 - Mietzner, Brun A1 - Steinberg, Pablo A1 - Pfeiffer, Andreas F. H. A1 - Ristow, Michael T1 - Induction of oxidative metabolism by mitochondrial frataxin inhibits cancer growth : Otto Warburg revisited N2 - More than 80 years ago Otto Warburg suggested that cancer might be caused by a decrease in mitochondrial energy metabolism paralleled by an increase in glycolytic flux. In later years, it was shown that cancer cells exhibit multiple alterations in mitochondrial content, structure, function, and activity. We have stably overexpressed the Friedreich ataxia-associated protein frataxin in several colon cancer cell lines. These cells have increased oxidative metabolism, as shown by concurrent increases in aconitase activity, mitochondrial membrane potential, cellular respiration, and ATP content. Consistent with Warburg's hypothesis, we found that frataxin-overexpressing cells also have decreased growth rates and increased population doubling times, show inhibited colony formation capacity in soft agar assays, and exhibit a reduced capacity for tumor formation when injected into nude mice. Furthermore, overexpression of frataxin leads to an increased phosphorylation of the tumor suppressor p38 mitogen-activated protein kinase, as well as decreased phosphorylation of extracellular signal-regulated kinase. Taken together, these results support the view that an increase in oxidative metabolism induced by mitochondrial frataxin may inhibit cancer growth in mammals Y1 - 2006 UR - http://www.jbc.org/content/281/2/977.full.pdf+html U6 - https://doi.org/10.1074/jbc.M511064200 ER - TY - JOUR A1 - Thierbach, René A1 - Drewes, Gunnar A1 - Fusser, Markus A1 - Wolfrum, Kathrin A1 - Epe, Bernd A1 - Ristow, Michael A1 - Steinberg, Pablo T1 - A role for iron-sulfur cluster proteins in DNA repair Y1 - 2009 UR - http://www.springerlink.com/content/100530 U6 - https://doi.org/10.1007/s00210-009-0404-1 SN - 0028-1298 ER - TY - JOUR A1 - Thierbach, René A1 - Drewes, Gunnar A1 - Fusser, Markus A1 - Voigt, Anja A1 - Kuhlow, Doreen A1 - Blume, Urte A1 - Schulz, Tim Julius A1 - Reiche, Carina A1 - Glatt, Hansruedi A1 - Epe, Bernd A1 - Steinberg, Pablo A1 - Ristow, Michael T1 - The Friedreich's ataxia protein frataxin modulates DNA base excision repair in prokaryotes and mammals N2 - DNA-repair mechanisms enable cells to maintain their genetic information by protecting it from mutations that may cause malignant growth. Recent evidence suggests that specific DNA-repair enzymes contain ISCs (iron-sulfur clusters). The nuclear-encoded protein frataxin is essential for the mitochondrial biosynthesis of ISCs. Frataxin deficiency causes a neurodegenerative disorder named Friedreich's ataxia in humans. Various types of cancer occurring at young age are associated with this disease, and hence with frataxin deficiency. Mice carrying a hepatocyte- specific disruption of the frataxin gene develop multiple liver tumours for unresolved reasons. In the present study, we show that frataxin deficiency in murine liver is associated with increased basal levels of oxidative DNA base damage. Accordingly, eukaryotic V79 fibroblasts overexpressing human frataxin show decreased basal levels of these modifications, while prokaryotic Salmonella enterica serotype Typhimurium TA 104 strains transformed with human frataxin show decreased mutation rates. The repair rates of oxidative DNA base modifications in V79 cells overexpressing frataxin were significantly higher than in control cells. Lastly, cleavage activity related to the ISC-independent repair enzyme 8-oxoguanine glycosylase was found to be unaltered by frataxin overexpression. These findings indicate that frataxin modulates DNA-repair mechanisms probably due to its impact on ISC-dependent repair proteins, linking mitochondrial dysfunction to DNA repair and tumour initiation. Y1 - 2010 UR - http://www.biochemj.org/bj/toc.htm U6 - https://doi.org/10.1042/Bj20101116 SN - 0264-6021 ER - TY - JOUR A1 - Thierbach, René A1 - Blume, Urte A1 - Wolfrum, K. A1 - Drewes, Gunnar A1 - Voigt, Anja A1 - Ristow, Michael A1 - Steinberg, Pablo T1 - Altered carbohydrate metabolism in a tumour developing knock-out mice model Y1 - 2010 UR - http://www.springerlink.com/content/100530 U6 - https://doi.org/10.1007/s00210-010-0508-7 SN - 0028-1298 ER - TY - JOUR A1 - Thierbach, Rene A1 - Florian, Simone A1 - Wolfrum, Katharina A1 - Voigt, Anja A1 - Drewes, Gunnar A1 - Blume, Urte A1 - Bannasch, Peter A1 - Ristow, Michael A1 - Steinberg, Pablo T1 - Specific alterations of carbohydrate metabolism are associated with hepatocarcinogenesis in mitochondrially impaired mice JF - Human molecular genetics N2 - Friedreich's ataxia is an inherited neurodegenerative disease caused by the reduced expression of the mitochondrially active protein frataxin. We have previously shown that mice with a hepatocyte-specific frataxin knockout (AlbFxn(-/-)) develop multiple hepatic tumors in later life. In the present study, hepatic carbohydrate metabolism in AlbFxn(-/-) mice at an early and late life stage was analyzed. In young (5-week-old) AlbFxn(-/-) mice hepatic ATP, glucose-6-phosphate and glycogen levels were found to be reduced by similar to 74, 80 and 88%, respectively, when compared with control animals. This pronounced ATP, G6P and glycogen depletion in the livers of young mice reverted in older animals: while half of the mice die before 30 weeks of age, the other half reaches 17 months of age and exhibits glycogen, G6P and ATP levels similar to those in age-matched controls. A key event in this respect seems to be the up-regulation of GLUT1, the predominant glucose transporter in fetal liver parenchyma, which became evident in AlbFxn(-/-) mice being 5-12 weeks of age. The most significant histological findings in animals being 17 or 22 months of age were the appearance of multiple clear cell, mixed cell and basophilic foci throughout the liver parenchyma as well as the development of hepatocellular adenomas and carcinomas. The hepatocarcinogenic process in AlbFxn 2/2 mice shows remarkable differences regarding carbohydrate metabolism alterations when compared with all other chemically and virally driven liver cancer models described up to now. Y1 - 2012 U6 - https://doi.org/10.1093/hmg/ddr499 SN - 0964-6906 VL - 21 IS - 3 SP - 656 EP - 663 PB - Oxford Univ. Press CY - Oxford ER -