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.