AB254. DU145: a naturally occurring cell with ATG5-independent alternative macroautophagy

Abstract

BackgroundAutophagy serves as a catabolic pathway to maintain cellular homeostasis. The role of autophagy in cancer cells is essential for supplying adequate substrates for proliferation and metabolism. Recent study identified DU145, different from LNCaP and PC-3 cells, was an autophagy-deficient cell line due to the deficiency of ATG5 protein. However, ATG5-independent alternative macroautophagy has already been discovered in ATG5 knockout mouse embryo fibroblasts. In this study, our goal is to discover whether DU145 is a naturally occurring cell with such an alternative autophagy.MethodsThe lipidation levels of microtubule-associated protein 1 light chain 3 (LC3) was detected by Western blotting. Cultured cells were transfected with the GFP-LC3 expressing plasmid and the fluorescence was viewed using confocal microscopy. Ultrastructure was observed using transmission electron microscopy. Immuno-electron microscopy was performed to determine the co-location of LC3 and autophagic vacuoles.ResultsUnder basal condition, the lipidation form of LC3, known as LC3-II, is detectable in both PC-3 and LNCaP cells. Stimulating autophagy with rapamycin or blocking autophagosome degradation with chloroquine increased the LC3-II levels in these cell lines. In contrast, LC3-II is constitutively undetectable in DU145 cells even treated with rapamycin or chloroquine. Similarly, the formation of autophagosome indicated by GFP-LC3 punctate was not easily observed in DU145 treated with rapamycin or chloroquine. Interestingly, we observed a great number of intracellular autophagic vacuoles under electron microscopy in all of three cell lines after starvation. Immuno-electron microscopy was applied with primary antibodies against LC3. Autophagic vacuoles labeled well with antibody against LC3-II in both PC-3 and LNCaP cells, rather than DU145 cells. These results suggested that DU145 is a naturally occurring cell with ATG5-independent autophagy.ConclusionsOur findings reveal that DU145 is a naturally occurring cell with an ATG5-independent alternative autophagy, which indicates a novel category of tumor cells may exists in prostate cancer patients.