J.C. Fernandez-Checa

OXIDATIVE DAMAGE OF MITOCHONDRIAL AND NUCLEAR DNA INDUCED BY IONIZING RADIATION IN HUMAN HEPATOBLASTOMA CELLS

PURPOSEnSince reactive oxygen species (ROS) act as mediators of radiation-induced cellular damage, the aim of our studies was to determine the effects of ionizing radiation on the regulation of hepatocellular reduced glutathione (GSH), survival and integrity of nuclear and mitochondrial DNA (mtDNA) in human hepatoblastoma cells (Hep G2) depleted of GSH prior to radiation.nnnMETHODS AND MATERIALSnGSH, oxidized glutathione (GSSG), and generation of ROS were determined in irradiated (50-500 cGy) Hep G2 cells. Clonogenic survival, nuclear DNA fragmentation, and integrity of mtDNA were assessed in cells depleted of GSH prior to radiation.nnnRESULTSnRadiation of Hep G2 cells (50-400 cGy) resulted in a dose-dependent generation of ROS, an effect accompanied by a decrease of reduced GSH, ranging from a 15% decrease for 50 cGy to a 25% decrease for 400 cGy and decreased GSH/GSSG from a ratio of 17 to a ratio of 7 for controls and from 16 to 6 for diethyl maleate (DEM)-treated cells. Depletion of GSH prior to radiation accentuated the increase of ROS by 40-50%. The depletion of GSH by radiation was apparent in different subcellular sites, being particularly significant in mitochondria. Furthermore, depletion of nuclear GSH to 50-60% of initial values prior to irradiation (400 cGy) resulted in DNA fragmentation and apoptosis. Consequently, the survival of Hep G2 to radiation was reduced from 25% of cells not depleted of GSH to 10% of GSH-depleted cells. Fitting the survival rate of cells as a function of GSH using a theoretical model confirmed cellular GSH as a key factor in determining intrinsic sensitivity of Hep G2 cells to radiation. mtDNA displayed an increased susceptibility to the radiation-induced loss of integrity compared to nuclear DNA, an effect that was potentiated by GSH depletion in mitochondria (10-15% intact mtDNA in GSH-depleted cells vs. 25-30% of repleted cells).nnnCONCLUSIONnGSH plays a critical protective role in maintaining nuclear and mtDNA functional integrity, determining the intrinsic radiosensitivity of Hep G2. Although the DNA repair is a complex process that is not yet completely understood, the protective role of GSH probably does not seem to involve the repair of classical DNA damage but may relate to modification of DNA damage dependent signaling.

Transcriptional regulation of the heavy subunit chain of γ‐glutamylcysteine synthetase by ionizing radiation

Since glutathione (GSH) protects against oxidative stress, we determined the regulation of cellular GSH by ionizing radiation in human hepatoblastoma cells, HepG2. The levels of GSH increased in irradiated HepG2 due to a greater γ‐glutamylcysteine synthetase (γ‐GCS) activity, which was paralleled by γ‐GCS heavy subunit chain (γ‐GCS‐HS) mRNA levels. Transcription of deletion constructs of the γ‐GCS‐HS promoter cloned in a reporter vector was associated with activator protein‐1 (AP‐1), consistent with the DNA binding of AP‐1 in nuclear extracts of irradiated HepG2. Hence, the transcriptional regulation of γ‐GCS by ionizing radiation emerges as an adaptive mechanism, which may be of significance to control the consequences of the oxidative stress induced by radiation.

P0299 : Sorafenib effect on mitochondrial function provides a target for increasing HCC therapy efficacy

Trabajo presentado en el 50th International Liver Congress, celebrado en Viena, Austria, del 22 al 26 de abril de 2015

P0293 : Targeting glucosylceramide synthase increases sorafenib efficacy and evades sorafenib resistance in HCC models

Trabajo presentado en el 50th International Liver Congress, celebrado en Viena, Austria, del 22 al 26 de abril de 2015

P0307 : Melatonin-induced apoptosis of HepG2 cells is enhanced by autophagy suppression

P0307 MELATONIN-INDUCED APOPTOSIS OF HepG2 CELLS IS ENHANCED BY AUTOPHAGY SUPPRESSION R. Ordonez, A. Fernandez, L. Martinez, S. Nunez, S. CarbajoPescador, A. Baulies, N. Prieto-Dominguez, C. Garcia-Ruiz, J.C. Fernandez-Checa, J.L. Mauriz, J. Gonzalez-Gallego. Institute of Biomedicine (IBIOMED), University of Leon (Spain), Leon, Centro de Investigacion Biomedica en Red: Enfermedades hepaticas y Digestivas (CIBERehd). Instituto de Salud Carlos III, Madrid, Department of Cell Death and Proliferation, Instituto Investigaciones Biomedicas de Barcelona, CSIC, Barcelona, and Liver Unit-Hospital Clinic-IDIBAPS, Barcelona, Spain E-mail: rordf@unileon.es

SREBP-2-mediated cholesterol accumulation regulates beta-amyloid–induced autophagy in APP/PS1/SREBP-2 mice

Comunicacion presentada en la Alzheimers Association 2013 International Conference, celebrada del 13 al 18 de julio de 2013 en Boston (Estados Unidos)

136 OVEREXPRESSION OF 2-OXOGLUTARATE CARRIER IN HEPATOCELLULAR CARCINOMA OVERCOMES CHOLESTEROL-MEDIATED MITOCHONDRIAL GSH DEPLETION TO EVADE CELL DEATH AND PROMOTE IN VIVO TUMOR GROWTH

Trabajo presentado al 46th Annual Meeting of the European Association for the Study of the Liver (EASL) celebrado en Berlin del 30 de marzo al 3 de abril de 2011.

APP/PS1 transgenic mice overexpressing SREBP2 as a new model to study the pathogenic role of cholesterol in Alzheimer's disease (AD)

Trabajo presentado a la Alzheimers Association 2011 International Conference on Alzheimers Disease (ICAD 2011) celebrada en Paris del 16 al 21 de julio de 2011.

95 CAVEOLIN-1 REGULATES MITOCHONRIAL CHOLESTEROL AND HEPATOCELLULAR SUSCEPTIBILITY TO TNF/FAS

Trabajo presentado al 45th Annual Meeting of the European Association for the Study of the Liver (EASL) celebrado en Viena del 14 al 18 de abril de 2010.

P4-184: Increased mitochondrial cholesterol enhances glial activation and neuronal oxidative stress induced by intracerebroventricular infusion of human beta-amyloid

Comunicacion presentada en la Alzheimers Association International Conference on Alzheimers Disease, celebrada del 26 al 31 de julio de 2008 en Chicago (Estados Unidos)