Promyelocytic HL60 Cells Express NADPH Oxidase and Are Excellent Targets in a Rapid Spectrophotometric Microplate Assay for Extracellular Superoxide

Abstract

A great number of drugs, toxicants, and growth factors induce the generation of intermediary reactive oxygen species (ROS). The human promyelocytic leukemia HL60 cell line differentiated along the macrophage or neutrophil lineage is a model system that is frequently used for the generation of ROS by various agents. As a primary source of ROS the superoxide anion produced by an enzymatic complex, NADPH oxidase, is well established. The present study shows that nondifferentiated HL60 cells contain NADPH oxidase and can be used as a model for the assessment of oxidant as well as antioxidant compounds. The expression of the multicomponent NADPH oxidase was demonstrated in nondifferentiated HL60 cells at the molecular level by detection of the mRNAs of the components gp91phox, p47phox, and p67phox as well as functionally by phorbol 12-myristate-13-acetate (PMA)-stimulated generation of superoxide, which was susceptible to inhibition by diphenyleneiodonium. The functional assay was performed using the cells in a log growth phase by adapting a standard microplate assay based on the classic superoxide dismutase-inhibitable reduction of cytochrome c. Validation of the microplate assay was carried out both with nonadherent differentiated HL60 cells and the adherent mouse monocyte-macrophage-like RAW 264.7 cell line, as well as with various compounds of oxidant (bleomycin sulfate, cis-diammineplatinum(II), camptothecin, TNF-alpha, IL-1 beta), nonoxidant (4 alpha-PMA, piracetam), and antioxidant (alpha-tocopherol, ascorbic acid) activity. In summary, we established a highly specific, reproducible and--with the aid of the nondifferentiated HL60 cell line--time-saving superoxide microplate assay as a valuable tool for the rapid screening of compounds for oxidative and antioxidative activity.

PVM/MA-shelled selol nanocapsules promote cell cycle arrest in A549 lung adenocarcinoma cells

“…The HL60 cell line is a cell model that is frequently used to access the oxidant or antioxidant potential of different compounds [34,35]. In the current study, Selol surprisingly did not induce an oxidative burst on HL60 cells (Figure 7(b)).…”

“…The ROS production was evaluated in A549 and HL60 cells [34,35]. The HL60 cells were cultured in RPMI (Roswell Park Memorial Institute medium) supplemented with 10% FBS and 1% antibiotic solution and were maintained at 37°C, 5% CO 2 and 80% humidity.…”

Nuclear Envelope Composition Determines the Ability of Neutrophil-type Cells to Passage through Micron-scale Constrictions

“…Respiratory Burst Assay-We determined superoxide radical production of day 4/ATRA-treated cells using luminolenhanced chemiluminescence (Diogenes reagent, National Diagnostics, Atlanta, GA) following stimulation by phorbol 12-myristate 13-acetate (25,26), as per the manufacturer's instructions. Cells were activated by the addition of phorbol 12-myristate 13-acetate (stock solution 1 mg/ml dimethyl sulfoxide (DMSO), Sigma) to a final concentration of 11 M; we recorded luminescence values after 30 min using a plate reader (SpectraMax M5).…”

New cellular tools reveal complex epithelial–mesenchymal interactions in hepatocarcinogenesis

“…Upon stimulation with PMA or LPS they released superoxide to the medium. Thus, the cell lines highly resemble B-cells, and exhibited some features known from macrophages such as phagocytosis and superoxide-production (Table 1; Parzefall et al, 2001;Teufelhofer et al, 2003).…”