Transcriptional activation of the gp91phox NADPH oxidase subunit by TPA inHL-60 cells

The exposure to epigenetic effecters capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflamma tion, tumor initiation, and promotion, The objective of this study was to e xamine the regulation of gg91phox, the catalytic subunit of the NADPH oxida se, and the kinetics of ROS production in promyelocytic leukemia HL-60 cell s induced with 12-O-tetradeconylphorbol-13-acetate (TPA), The treatment of HL-60 cells with TPA (0.1 muM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigen in and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholor ofluorescin (DCFH). Whereas higher concentrations (1.0 muM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 muM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increas e in chemiluminescence. In cells treated with TPA, the burst in ROS at 48 h was preceded by accumulation at 12 h of gp91phox (8.8-fold) and p47phox mR NA (threefold), whereas untreated cells contained steady-state levels of bo th transcripts, Timecourse experiments with actinomycin D to inhibit transc ription revealed that TPA did not improve the stability of gp91phox. In tra nsient transfections, luciferase reporter activity directed from a 1.5-kb g p91phox promoter fragment was enhanced threefold upon treatment with TPA fo r 24 h, We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.