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(Journal of Leukocyte Biology. 2001;69:161-168.)
© 2001 by Society for Leukocyte Biology

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

David J. Samuelson*,{dagger}, Marianne-B Powell{ddagger}, Maria Lluria-Prevatt* and Donato F. Romagnolo*,{dagger},§,||

* Interdisciplinary Cancer Biology Program
§ Arizona Cancer Center
|| Southwest Environmental Health Sciences Center
{dagger} Laboratory of Mammary Gland Biology, Department of Nutritional Sciences, The University of Arizona, Tucson, Arizona
{ddagger} Division of Radiation Biology, Stanford University, Stanford, California

Correspondence: Donato F. Romagnolo, 303 Shantz Bldg., The University of Arizona, Tucson, AZ 85721-0038. E-mail: donato{at}ag.arizona.edu

The exposure to epigenetic effectors capable of inducing copious production of reactive oxygen species (ROS) has been associated with chronic inflammation, tumor initiation, and promotion. The objective of this study was to examine the regulation of gp91phox, the catalytic subunit of the NADPH oxidase, and the kinetics of ROS production in promyelocytic leukemia HL-60 cells induced with 12-O-tetradeconylphorbol-13-acetate (TPA). The treatment of HL-60 cells with TPA (0.1 µM) induced cellular differentiation, which was followed after 48 h by a tenfold increase in chemiluminescence from lucigenin and a 2.5-fold increase in the intracellular oxidation of 2',7'-dicholorofluorescin (DCFH). Whereas higher concentrations (1.0 µM) of TPA did not stimulate further ROS production, repeated stimulation with 0.1 µM TPA of differentiated cells induced a modest (1.2-fold) but rapid (15 min) increase 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 mRNA (threefold), whereas untreated cells contained steady-state levels of both transcripts. Time-course experiments with actinomycin D to inhibit transcription revealed that TPA did not improve the stability of gp91phox. In transient transfections, luciferase reporter activity directed from a 1.5-kb gp91phox promoter fragment was enhanced threefold upon treatment with TPA for 24 h. We conclude that TPA can commit HL-60 cells to differentiation and elicit transcription from the proximal gp91phox promoter.




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