EFFECTS OF AUTOPHAGY MODULATION BY HYDROXYCHLOROQUINE ON GROWTH ARREST-SPECIFIC P20K PROTEIN LEVELS IN CHICKEN EMBRYO FIBROBLASTS

Abstract

Autophagy and reversible growth arrest are two cellular processes that play crucial roles in unfavorable conditions. Autophagy involves the degradation and recycling of damaged or unnecessary cellular components. Reversible growth arrest is entered by cells under unfavorable conditions for proliferation, such as contact inhibition, starvation, and hypoxia. This study investigates the potential relationship between autophagy and the regulation of growth arrest-specific (GAS) proteins. To address this question, this study examines the impact of inhibiting autophagy using hydroxychloroquine (HCQ) on the accumulation of the growth arrest-specific proteins, including the p20K lipocalin, in chicken embryo fibroblasts (CEFs). Results from western blot analysis of ATG8 homologs and immunofluorescence microscopy of the dual-reporter protein mCherry-EGFP-LC3B confirmed HCQ’s inhibitory effects on autophagy in CEFs. The western blot analysis showed that inhibition of autophagy leads to the accumulation of GAS proteins, particularly p20K and CutA, as well as the endoplasmic reticulum stress marker CHOP. The presence of p20K in HCQ-treated cells was visualized by immunofluorescence microscopy. Interestingly, levels of the p20K increase after 6 hours of HCQ treatment, whereas CutA levels increase rapidly after just one hour of treatment. Conversely, the down-regulation of p20K does not affect the activation of autophagy-related proteins GABARAP and LC3, suggesting the relationship between autophagy and GAS protein is not reciprocal. Furthermore, proliferation assays demonstrate a significant difference in cell counts between HCQ-treated CEFs and control CEFs, raising the possibility that the effects of HCQ on growth arrest-specific genes are indirect. In addition, we provided evidence that two members of the ATG8 family, GABARAP and LC3, are localized in different autophagic vesicles. In conclusion, this study clarified the relationship between different biological processes and provides insights into cell homeostasis during unfavorable conditions for proliferation.