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Articles in PresS, published online ahead of print January 2, 2002
Am J Physiol Cell Physiol, 10.1152/ajpcell.00496.2001
Submitted on October 16, 2001
Accepted on December 17, 2001
1 Internal Medicine, Carolinas Medical Center, Charlotte, NC 28232, USA
2 Division of Respiratory, Critical Care and Occupational (Pulmonary) Medicine, University of Utah, Salt Lake City, UT 84132, USA
3 Veterinary Molecular Biology, Montana State University, Bozeman, MT 59717, USA
4 Pharmacology and Cancer Biology, Duke University, Durham, NC 27710, USA
* To whom correspondence should be addressed. E-mail: tkennedy{at}carolinas.org.
Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-
B (NF-B). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-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 the 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 were 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. The flavoprotein inhibitor diphenylene iodonium also inhibited constitutive DNA binding of nuclear protein to the NF-B and cyclic-AMP response element consensus oligonucleotides, without affecting DNA binding activity to AP-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.
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