title: Autophagy is required during cycling hypoxia to lower production of reactive oxygen species.
abstract: OBJECTIVE
  Human tumors are characterized by the presence of cells that experience periodic episodes of hypoxia followed by reoxygenation. These cells are exposed to reactive oxygen species (ROS) upon reoxygenation and require adaptation to this stress by lowering ROS production or enhancing ROS-clearance for their survival. We hypothesized that autophagy, a lysosomal degradation pathway, may be involved in reducing ROS during periodic hypoxia through removal of ROS producing species.
  
  
  METHODS
  Human tumor cells (MCF-7, HT29, U373) were exposed to cycles of hypoxia (O(2)<0.02%) and reoxygenation in the absence or presence of the autophagy inhibitor chloroquine (CQ). Clonogenic survival, ROS production and mitochondrial-DNA content were assessed. In addition, A549 cells overexpressing wild-type or K63-mutated ubiquitin (K63R) were analyzed for ROS production.
  
  
  RESULTS
  Our data indicate that CQ treatment sensitizes cells to cycling hypoxia, due to increased production of ROS, associated with an incapacity to reduce mitochondrial content. Addition of the ROS-scavenger N-acetyl-cysteine increased cell viability and neutralized CQ-effects. Additionally, genetic prevention of K63-linked ubiquitin chains that are required for the removal of toxic protein aggregates by autophagy, resulted in increased ROS production.
  
  
  CONCLUSIONS
  Inhibition of autophagy substantially increases cell death induced by cycling hypoxia through increased ROS production, providing an opportunity to decrease the hypoxic fraction within tumors and enhance tumor therapy.
authors.affiliation: Maastricht Radiation Oncology (MaastRo) Lab, GROW-School for Oncology and Developmental Biology, University of Maastricht, The Netherlands. Kasper.Rouschop@maastro.unimaas.nl
authors.forename: Kasper M A
authors.initials: KM
authors.lastname: Rouschop
authors.forename: Chantal H M A
authors.initials: CH
authors.lastname: Ramaekers
authors.forename: Marco B E
authors.initials: MB
authors.lastname: Schaaf
authors.forename: Tom G H
authors.initials: TG
authors.lastname: Keulers
authors.forename: Kim G M
authors.initials: KG
authors.lastname: Savelkouls
authors.forename: Philippe
authors.initials: P
authors.lastname: Lambin
authors.forename: Marianne
authors.initials: M
authors.lastname: Koritzinsky
authors.forename: Bradly G
authors.initials: BG
authors.lastname: Wouters
mesh_terms: D000704:Analysis of Variance; D001343:Autophagy / Q000187:drug effects / Q000502:physiology*; D015153:Blotting, Western; D016923:Cell Death / Q000187:drug effects / Q000502:physiology; D045744:Cell Line, Tumor; D002470:Cell Survival / Q000187:drug effects / Q000502:physiology; D002738:Chloroquine / Q000494:pharmacology*; D004272:DNA, Mitochondrial / Q000187:drug effects / Q000378:metabolism*; D005434:Flow Cytometry; D006801:Humans; D000860:Hypoxia; D018384:Oxidative Stress / Q000187:drug effects / Q000502:physiology; D011336:Probability; D017382:Reactive Oxygen Species / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
chemical_list: D004272:DNA, Mitochondrial; D017382:Reactive Oxygen Species; D002738:Chloroquine