Ashik Asvin Patel

By Ashik Asvin Patel and Gary Warnes

Cell cycle-arresting drugs, thapsigargin (Tg) and chloroquine (CQ), are employed to study endoplasmic reticulum (ER) stress and the autophagic process using cell lines without measuring the cell cycle of such cells. The potential cell cycle-dependent aspect of such processes in cell lines may impact upon the degree of ER stress and autophagy measured. ER stress is known to be caused by a build-up of misfolded proteins within the ER, which may then undergo ER phagy or reticulophagy. The cell cycle-dependent nature of all these processes is not well studied, so we investigated ER stress and autophagy by use of a combination of flow cytometric assays. These included cell cycle-dependent measurement of reticulophagy, misfolded protein levels and autophagic marker LC3-II in K562 and Jurkat cells. ER stress-inducing drug Tg caused significant reticulophagy in both cell types. This was cell cycle dependent in K562 cells only, with proliferating cells undergoing more reticulophagy. In contrast, autophagy-initiating drug CQ caused reticulophagy at higher doses in Jurkat cells, whereas K562 cells showed a cell cycle-dependent elongation of the ER, which was less pronounced in proliferating cells. The level of cellular misfolded protein in response to both drugs was high in K562 cells when either undergoing reticulophagy or elongation in a non-cell cycle-dependent manner, whereas the misfolded protein levels in Jurkat cells in response to both drugs were lower than those observed in K562 cells. Both cell lines employed in this study showed no increase of LC3-II above controls in response to Tg treatment. However, CQ induced a cell cycle-dependent increase of LC3-II in both cell types. Thus, the type of cell employed and the cell cycle dependent modulation of thebiological processes involved in ER stress and autophagy should be considered when designing studies in ER stress and autophagy.

Part of the book: Autophagy in Current Trends in Cellular Physiology and Pathology