title: Canonical autophagy does not contribute to cellular radioresistance.
abstract: BACKGROUND
  (Pre)clinical studies indicate that autophagy inhibition increases response to anti-cancer therapies. Although promising, due to contradicting reports, it remains unclear if radiation therapy changes autophagy activity and if autophagy inhibition changes the cellular intrinsic radiosensitivity. Discrepancies may result from different assays and models through off-target effects and influencing other signaling routes. In this study, we directly compared the effects of genetic and pharmacological inhibition of autophagy after irradiation in human cancer cell lines.
  
  
  METHODS
  Changes in autophagy activity after ionizing radiation (IR) were assessed by flux analysis in eight cell lines. Clonogenic survival, DNA damage (COMET-assay) and H2AX phosphorylation were assessed after chloroquine or 3-methyladenine pretreatment and after ATG7 or LC3b knockdown.
  
  
  RESULTS
  IR failed to induce autophagy and chloroquine failed to change intrinsic radiosensitivity of cells. Interestingly, 3-methyladenine and ATG7- or LC3b-deficiency sensitized cancer cells to irradiation. Surprisingly, the radiosensitizing effect of 3-methyladenine was also observed in ATG7 and LC3b deficient cells and was associated with attenuated γ-H2AX formation and DNA damage repair.
  
  
  CONCLUSIONS
  Our data demonstrate that the anti-tumor effects of chloroquine are independent of changes in intrinsic radioresistance. Furthermore, ATG7 and LC3b support radioresistance independent of canonical autophagy that involves lysosomal degradation.
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Marco B E
authors.initials: MB
authors.lastname: Schaaf
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Barry
authors.initials: B
authors.lastname: Jutten
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Tom G
authors.initials: TG
authors.lastname: Keulers
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Kim G M
authors.initials: KG
authors.lastname: Savelkouls
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Hanneke J M
authors.initials: HJ
authors.lastname: Peeters
authors.affiliation: Department of Toxicogenomics, The Netherlands.
authors.forename: Twan
authors.initials: T
authors.lastname: van den Beucken
authors.affiliation: Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, The Netherlands.
authors.forename: Frederik-Jan
authors.initials: FJ
authors.lastname: van Schooten
authors.affiliation: Department of Toxicology, NUTRIM School for Nutrition, Toxicology and Metabolism, Maastricht University, The Netherlands.
authors.forename: Roger W
authors.initials: RW
authors.lastname: Godschalk
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands.
authors.forename: Marc
authors.initials: M
authors.lastname: Vooijs
authors.affiliation: Department of Radiation Oncology (Maastro Lab), GROW School for Oncology & Developmental Biology, Maastricht University Medical Center+, The Netherlands. Electronic address: Kasper.Rouschop@maastrichtuniversity.nl.
authors.forename: Kasper M A
authors.initials: KM
authors.lastname: Rouschop
mesh_terms: D000225:Adenine / Q000031:analogs & derivatives / Q000494:pharmacology; D001343:Autophagy / Q000187:drug effects; D045744:Cell Line, Tumor; D002738:Chloroquine / Q000494:pharmacology; D004260:DNA Repair / Q000187:drug effects; D006801:Humans; D010766:Phosphorylation; D011836:Radiation Tolerance / Q000235:genetics; D011839:Radiation, Ionizing; D011838:Radiation-Sensitizing Agents / Q000494:pharmacology; D015398:Signal Transduction / Q000187:drug effects
chemical_list: D011838:Radiation-Sensitizing Agents; C025946:3-methyladenine; D002738:Chloroquine; D000225:Adenine
keywords: 3-Methyladenine; ATG7; Autophagy; Chloroquine; Ionizing radiation; LC3b