A Novel Drug Discovery Assay For Analyzing Autophagy In Living Cells

Proceedings: AACR 102nd Annual Meeting 2011‐‐ Apr 2‐6, 2011; Orlando, FL Autophagy is a normal degradative pathway that involves the sequestration of entire organelles, protein complexes, and misfolded proteins in a membrane vacuole called the autophagosome. The autophagosomal vesicle is subsequently delivered to the lysosome where it is degraded into its essential constituents and recycled back to the cytoplasm. Autophagy plays an important role in diverse biological events, not only during conditions of nutrient limitation, but it also functions in tumor suppression, the immune response, and the prevention of certain types of neurodegeneration. However, experimental methods to monitor this process in mammalian cells are limited due to lack of autophagic markers. Recently, LC3, a mammalian homologue of the ubiquitin-like (UBL) protein Atg8, has been used as a specific marker of autophagosomes in mammals. However, current methods to quantify autophagic activity using LC3 are time-consuming and labor-intensive. We have developed and validated a novel cell-based autophagy assay using a 488 nm-excitable green-emitting fluorescent probe to highlight the various vacuolar components of the autophagy pathway. We demonstrated that the accumulation of autophagy probe was specifically induced by amino acid deprivation and was inhibited by 3-methlyadenine, a classical inhibitor of the autophagic pathway. Furthermore, a population of this dye-labeled vesicle co-localizes with LC3. We have validated this fluorescent probe with a wide range of conditions known to modulate autophagy pathways. The assay is compatible with flow cytometry, allowing, for the first time, easy quantitation of autophagy. This assay enables kinetic analysis of the autophagy pathway and is able to distinguish between increases in autophagic flux vs. autophagic vacuole accumulation. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 3774. doi:10.1158/1538-7445.AM2011-3774