Overexpression of ATG5 Gene Makes Granulocyte-Like HL-60 Susceptible to Release Reactive Oxygen Species

• Published in: International journal of molecular sciences 2020-07, Vol.21 (15), p.5194
• Main Authors: Mroczek, Agnieszka, Cieloch, Adrianna, Manda-Handzlik, Aneta, Kuźmicka, Weronika, Muchowicz, Angelika, Wachowska, Małgorzata
• Format: Article
• Language: English
• Subjects: Apoptosis; Apoptosis - drug effects; ATG5; Autophagy; Autophagy - genetics; Autophagy-Related Protein 5 - genetics; Autophagy-Related Protein 5 - metabolism; Cell differentiation; Cell Differentiation - drug effects; Cell proliferation; Cell Proliferation - drug effects; Change detection; Deoxyribonucleic acid; Differentiation; Dimethylformamide - pharmacology; DNA; Flow Cytometry; Fluorescence; Fluorimetry; Fluorometry; Gene expression; Genetic modification; Granulocytes; Granulocytes - metabolism; HL-60 Cells; Humans; Immune system; Leukemia; Leukemia, Promyelocytic, Acute - genetics; Leukemia, Promyelocytic, Acute - metabolism; Leukocytes (granulocytic); Leukocytes (neutrophilic); Microtubule-Associated Proteins - metabolism; Neutrophils; Neutrophils - metabolism; neutrophils functions; Pathogens; Phagocytosis; Phagocytosis - drug effects; promyelocytic leukemia cells (HL-60); Promyeloid leukemia; Proteins; Reactive oxygen species; Reactive Oxygen Species - metabolism; Retinoic acid; Tretinoin - pharmacology; Up-Regulation
• ISSN: 1422-0067; 1661-6596; 1422-0067
• DOI: 10.3390/ijms21155194

Neutrophils represent the first line of defense against pathogens using various strategies, such as phagocytosis, production of reactive oxygen species (ROS) and neutrophil extracellular traps (NETs) formation. Recently, an autophagy-independent role of autophagy related ( ) gene 5 in immune cells, including neutrophils, was emphasized. Our aim was to investigate the role of ATG5 protein in neutrophils' antimicrobial functions, proliferation and apoptosis. To this end, we used genetically modified human promyelocytic leukemia (HL-60) cells overexpressing ATG5, differentiated toward granulocyte-like cells with all-trans retinoic acid (ATRA) and dimethylformamide. The level of differentiation, phagocytosis, proliferation and apoptosis were determined by flow cytometry. ROS production and NETs release was assessed by fluorometry and fluorescent microscopy. gene expression was evaluated by real-time PCR, whereas the protein level of ATG5 and LC3-II was determined by Western blot. We did not observe the induction of autophagy in differentiated HL-60 cells overexpressing ATG5. The increased expression of ATG5 affects the differentiation of HL-60 cells with ATRA, ROS production and phagocytosis. However, we did not detect changes in NETs release. Moreover, ATG5 protects differentiated HL-60 cells from apoptosis but does not cause changes in proliferation rate.