No abstract

The mechanism of neutrophil extracellular trap cell death (NETosis), a regulated cell death pathway relevant to infection, autoimmunity and sepsis, is not completely known. The reason for this, at least in part, is the lack of an in vitro system that recapitulates the NETosis pathway using established human cell lines. We show that exposure of a human promyelocytic leukemia cell line HL-60 to the glycosyltransferase inhibitor tunicamycin (TM) resulted in extrusion of decompacted genomic DNAs to extracellular space, morphologically similar to NETs. Immunostaining using antibodies against NET marker proteins and bacterial trapping assay showed biochemical similarities between the TM-induced extracellular DNA structures and NETs. The NET-like structures were also generated on exposure of TM to other myeloid cell lines, such as U937 and THP-1. Thus, our findings provide an experimental setting to induce NET-like structures using cultured human myeloid cell lines, which may help our understanding of the regulation and function of NETosis.

Human myeloid HL‐60 cells are usually cultured in suspension in medium containing 5% to 10% fetal bovine serum (FBS) and thus are often difficult to adhere to a coverslip. In this unit, we describe how removal of FBS from the culture medium facilitates adhesion of HL‐60 cells to coverslips. Importantly, HL‐60 cells that adhere to the coverslips immersed in FBS‐free medium can be immobilized in situ by conventional chemical fixatives and thus permeabilized for probing cellular structures using specific dyes and/or reagents, followed by microscopic observation. All‐trans‐retinoic‐acid‐exposed differentiated HL‐60 cells, which have properties similar to neutrophils, can also adhere efficiently to coverslips in FBS‐free medium. Because the procedure is not complex and special equipment is not required, the simplicity and cost effectiveness of this FBS‐free cell adhesion protocol may be beneficial to researchers who are interested in assessing the structure and function of suspension cells using microscopy. © 2015 by John Wiley & Sons, Inc.

Small ubiquitin-related modifier (SUMO) is a highly conserved protein that is covalently attached to target proteins. This posttranslational modification, designated SUMOylation, is a major protein-conjugation-driven strategy designed to regulate structure and function of cellular proteins. SUMOylation consists of an enzymatic cascade involving the E1-activating enzyme and the E2-conjugating enzyme. The SUMO-E1 enzyme consists of two subunits, a heterodimer of activation of Smt3p 1 (Aos1) and ubiquitin activating enzyme 2 (Uba2), which resembles the N- and C-terminal halves of ubiquitin E1 (Uba1). Herein, we describe the rational design of a single polypeptide version of SUMO-E1, a chimera of mouse Aos1 and Uba2 subunits, termed mAU, in which the functional domains appear to be arranged in a fashion similar to Uba1. We also describe the construction of a mAU plasmid for expression in a baculovirus-insect cell system and present an in situ SUMOylation assay using the recombinant mAU. Our results showed that mAU has SUMO-E1 activity, thereby indicating that mAU can be expressed in baculovirus-insect cells and represents a suitable source of SUMO-E1.

We previously reported that the nucleoside antibiotic tunicamycin (TN), a protein glycosylation inhibitor triggering unfolded protein response (UPR), induced neutrophil extracellular trap-osis (NETosis)-like cellular suicide and, thus, discharged genomic DNA fibers to extracellular spaces in a range of human myeloid cell lines under serum-free conditions. In this study, we further evaluated the effect of TN on human promyelocytic leukemia HL-60 cells using time-lapse microscopy. Our assay revealed a previously unappreciated early event induced by TN-exposure, in which, at 30-60 min after TN addition, the cells extruded their nuclei into the extracellular space, followed by discharge of DNA fibers to form NET-like structures. Intriguingly, neither nuclear extrusion nor DNA discharge was observed when cells were exposed to inducers of UPR, such as brefeldin A, thapsigargin, or dithiothreitol. Our findings revealed novel nuclear dynamics during TN-induced NETosis-like cellular suicide in HL-60 cells and suggested that the toxicological effect of TN on nuclear extrusion and DNA discharge was not a simple UPR.