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Cell Death Triggered by the Autophagy Inhibitory Drug 3-Methyladenine in Growing Conditions Proceeds With DNA Damage Chicote, Javier Yuste, Victor José Boix, Jacint Ribas, Judit 3-methyladenine Autophagy inhibitor Basal autophagy Ɣ-H2A.X Apoptosis Macroautophagy (hereafter autophagy) is a multistep intracellular catabolic process with pleiotropic implications in cell fate. Attending to its activation, autophagy can be classified into inducible or constitutive. Constitutive, or basal autophagy, unfolds under nutrient-replete conditions to maintain the cellular homeostasis. Autophagy inhibitory drugs are powerful tools to interrogate the role of autophagy and its consequences on cell fate. However, 3-methyladenine and various of these compounds present an intrinsic capacity to trigger cell death, for instance the broadly-employed 3-methyladenine. To elucidate whether the inhibition of basal autophagy is causative of cell demise, we have employed several representative compounds acting at different phases of the autophagic process: initiation (SBI0206965 and MHY1485), nucleation (3-methyladenine, SAR405, Spautin-1 and Cpd18), and completion (Bafilomycin A and Chloroquine). These compounds inhibited the basal autophagy of MEF cultures in growing conditions. Among them, 3-methyladenine, SBI-0206965, Chloroquine, and Bafilomycin A triggered BAX- and/or BAK-dependent cytotoxicity and caspase activation. 3-methyladenine was the only compound to induce a consistent and abrupt decrease in cell viability across a series of ontologically unrelated human cell lines. 3-methyladenine-induced cytotoxicity was not driven by the inhibition of the AKT/mTOR axis. Autophagy-deficient Fip200-/- MEFs displayed an increased sensitivity to activate caspases and to undergo cell death in response to 3-methyladenine. The cytotoxicity induced by 3-methyladenine correlated with a massive DNA damage, as shown by γ -H2A.X. This genotoxicity was observed at 10 mM 3-methyladenine, the usual concentration to inhibit autophagy and was maximized in Fip200-/- MEFs. In sum, our results suggest that, in growing conditions, autophagy acts as a protective mechanism to diminish the intrinsic cytotoxicity of 3-methyladenine. However, when the cellular stress exerted by 3-methyladenine surpasses the protective effect of basal autophagy, caspase activation and DNA damage compromise the cell viability. 2020 Article https://ddd.uab.cat/record/253081 urn:10.3389/fphar.2020.580343 urn:oai:ddd.uab.cat:253081 urn:articleid:16639812v11 urn:pmcid:PMC7593545 urn:pmc-uid:7593545 urn:pmid:33178023 urn:oai:pubmedcentral.nih.gov:7593545 urn:oai:egreta.uab.cat:publications/7e253b9a-65be-4001-98c9-6c5fe43a24f6 urn:scopus_id:85094571451 eng Agencia Estatal de Investigación SAF2016-78657-R Frontiers in Pharmacology ; Vol. 11 (october 2020) open access Aquest document està subjecte a una llicència d'ús Creative Commons. Es permet la reproducció total o parcial, la distribució, la comunicació pública de l'obra i la creació d'obres derivades, fins i tot amb finalitats comercials, sempre i quan es reconegui l'autoria de l'obra original. https://creativecommons.org/licenses/by/4.0/ application/pdf