Malignant 
melanoma cells spontaneously generate 
reactive oxygen species (ROS) that promote constitutive activation of the 
transcription factor nuclear factor-kB (NF-kB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kB activation and suppress 
cell proliferation, 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. 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 91phox homolog 
NOX4 were demon-strated 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. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive 
DNA binding of 
nuclear protein to the NF-kB and cyclic-AMP 
response element consensus oligonucleotides, without affecting 
DNA binding activity to AP-1 or OCT-1.