Regulation of nad(p)h oxidase growth and transcription in melanoma cells

Abstract

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 p22, gp91and p67components of the human phagocyte NAD(P)H oxidase, and the 91homolog NOXwere demon-strated in melanomas by RT-PCR and sequencing, and protein product for both p22and gp91were detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22and NOX. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22and NOX. 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-.