Suppression of autophagy enhances the cytotoxicity of the DNA-damaging aromatic amine p-anilinoaniline.
p-Anilinoaniline (pAA) is an aromatic amine that is widely used in hair dying applications. It is also a metabolite of metanil yellow, an azo dye that is commonly used as a food coloring agent. Concentrations of pAA between 10 and 25 microM were cytostatic to cultures of the normal human mammary epithelia cell line MCF10A. Concentrations >or=50 microM were cytotoxic. Cytostatic concentrations induced transient G(1) and S cell cycle phase arrests; whereas cytotoxic concentrations induced protracted arrests. Cytotoxic concentrations of pAA caused DNA damage, as monitored by the alkaline single-cell gel electrophoresis (Comet) assay, and morphological changes consistent with cells undergoing apoptosis and/or autophagy. Enzymatic and western blot analyses, and binding analyses of fluorescent labeled VAD-FMK, suggested that caspase family members were activated by pAA. Western blot analyses documented the conversion of LC3-I to LC3-II, a post-translational modification involved in the development of the autophagosome. Suppression of autophagosome formation, via knockdown of ATG7 with shRNA, prevented pAA-induced vacuolization, enhanced the activation of pro-caspase-3, and increased susceptibility of ATG7-deficient cells to the cytostatic and cytotoxic activities of markedly lower concentrations of pAA. Cells stably transfected with a nonsense shRNA behaved like parental MCF10A cells. Collectively, these data suggest that MCF10A cultures undergo autophagy as a pro-survival response to concentrations of pAA sufficient to induce DNA damage.
Elliott
Althea
A
Institute of Environmental Health Sciences, Wayne State University, Detroit, MI 48201, USA.
Reiners
John J
JJ
D000409:Alanine / Q000031:analogs & derivatives* / Q000633:toxicity; D024322:Amino Acids, Aromatic / Q000633:toxicity; D017209:Apoptosis / Q000187:drug effects / Q000235:genetics; D001343:Autophagy / Q000187:drug effects* / Q000235:genetics; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D004249:DNA Damage / Q000187:drug effects* / Q000502:physiology; D004305:Dose-Response Relationship, Drug; D004847:Epithelial Cells / Q000187:drug effects / Q000473:pathology / Q000502:physiology; D006801:Humans
D024322:Amino Acids, Aromatic; C078798:3-(phenylamino)alanine; D000409:Alanine