Autophagy pathways activated in response to PDT contribute to cell resistance against ROS damage.
Bottom Line: Paradoxically, genetic loss of MA improved clearance of oxidized proteins and reduced photokilling.We found that up-regulation of chaperone-mediated autophagy (CMA) in unstressed Atg(-/-) cells compensated for MA loss and increased cellular resistance to PDT.These results disclose a stress-specific recruitment of autophagy pathways with cytoprotective function and unravel CMA as the dominant defence mechanism against PDT.
Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.Show MeSH
Related in: MedlinePlus
Mentions: Western blot analysis revealed that PDT-treatment of different cancer and immortalized cell lines evoked a progressive conversion of the cytosolic LC3-I into its lipidated form LC3-II, a specific biochemical marker of MA. This is as also visualized by the redistribution of GFP-tagged LC3 from a diffuse (cytosolic LC3-I) into a dotted pattern (LC3-II accumulating in autophagosomal membranes) (Fig. 1A and B). In line with these results, ultrastructural analysis of PDT-treated cells via TEM showed a clear vacuolization of the cytoplasm (Fig. 1C). To clarify whether accumulation of LC3-II in our paradigm reflects stimulation of the ON-rate (stimulation of autophagic degradation) of MA or results from a ROS-mediated inhibition of lysosomal function, we performed a flux-analysis using MEFs stably expressing GFP-LC3 . Addition of BafA1, an inhibitor of autophagosome degradation, increased the detection of LC3-II after treatment and inhibited the release of ‘free GFP’, a hallmark of autophagosome degradation (Fig. 1D). This indicates that PDT stimulates MA flux.
Affiliation: Cell Death Research and Therapy Laboratory, Department of Molecular Cell Biology, Faculty of Medicine, Katholieke Universiteit Leuven, Leuven, Belgium.