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Am J Physiol Cell Physiol 282: C1212-C1224, 2002. First published January 2, 2002; doi:10.1152/ajpcell.00496.2001
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Vol. 282, Issue 6, C1212-C1224, June 2002

An NAD(P)H oxidase regulates growth and transcription in melanoma cells

Sukhdev S. Brar1, Thomas P. Kennedy1, Anne B. Sturrock2, Thomas P. Huecksteadt2, Mark T. Quinn3, A. Richard Whorton4, and John R. Hoidal2

1 Departments of Internal Medicine and Cannon Research Center, Carolinas Medical Center, Charlotte, North Carolina 28232; 2 Division of Respiratory, Critical Care and Occupational (Pulmonary) Medicine, University of Utah, Salt Lake City, Utah 84132; 3 Department of Veterinary Molecular Biology, Montana State University, Bozeman, Montana 59717; and 4 Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, North Carolina 27710

Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappa B (NF-kappa B). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappa B activation and suppress cell proliferation (11), the nature of the enzyme responsible for ROS production in melanoma cells has not been determined. To address this issue, we now have characterized the source of ROS production in melanoma cells. We report that ROS are generated by isolated, cytosol-free melanoma plasma membranes, with inhibition by NAD(P)H oxidase inhibitors. The p22phox, gp91phox, and p67phox components of the human phagocyte NAD(P)H oxidase and the gp91phox homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22phox and gp91phox was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22phox and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22phox and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappa B and cAMP-response element consensus oligonucleotides, without affecting DNA binding activity to activator protein-1 or OCT-1. This suggests that ROS generated in autocrine fashion by an NAD(P)H oxidase may play a role in signaling malignant melanoma growth.

superoxide anion; diphenylene iodonium; p22phox; gp91phox; p67phox; NOX1; NOX4; nuclear factor-kappa B; cAMP response element; dicumarol


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