An NAD(P)H oxidase regulates growth and transcription in melanoma cells.
Malignant melanoma cells spontaneously generate reactive oxygen species (ROS) that promote constitutive activation of the transcription factor nuclear factor-kappaB (NF-kappaB). Although antioxidants and inhibitors of NAD(P)H oxidases significantly reduce constitutive NF-kappaB 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 p22(phox), gp91(phox), and p67(phox) components of the human phagocyte NAD(P)H oxidase and the gp91(phox) homolog NOX4 were demonstrated in melanomas by RT-PCR and sequencing, and protein product for both p22(phox) and gp91(phox) was detected in cell membranes by immunoassay. Normal human epidermal melanocytes expressed only p22(phox) and NOX4. Melanoma proliferation was reduced by NAD(P)H oxidase inhibitors and by transfection of antisense but not sense oligonucleotides for p22(phox) and NOX4. Also, the flavoprotein inhibitor diphenylene iodonium inhibited constitutive DNA binding of nuclear protein to the NF-kappaB 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.
Brar
Sukhdev S
SS
Department of Internal Medicine, Carolinas Medical Center, Charlotte, North Carolina 28232, USA.
Kennedy
Thomas P
TP
Sturrock
Anne B
AB
Huecksteadt
Thomas P
TP
Quinn
Mark T
MT
Whorton
A Richard
AR
Hoidal
John R
JR
D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D007424:Intracellular Fluid / Q000378:metabolism; D008544:Melanocytes / Q000166:cytology / Q000378:metabolism; D008546:Melanoma, Experimental / Q000378:metabolism* / Q000473:pathology; D008562:Membrane Glycoproteins / Q000235:genetics / Q000378:metabolism; D026901:Membrane Transport Proteins; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D009252:NADPH Dehydrogenase / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000096:biosynthesis; D009861:Onium Compounds / Q000494:pharmacology; D010750:Phosphoproteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism; D016133:Polymerase Chain Reaction; D021122:Protein Subunits; D012333:RNA, Messenger / Q000037:antagonists & inhibitors / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D020218:Response Elements / Q000502:physiology; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic
D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D016328:NF-kappa B; D009861:Onium Compounds; D010750:Phosphoproteins; D021122:Protein Subunits; D012333:RNA, Messenger; D017382:Reactive Oxygen Species; D014157:Transcription Factors; C007517:diphenyleneiodonium; D009247:NADH, NADPH Oxidoreductases; C056558:CYBB protein, human; D000074662:NADPH Oxidase 2; D000074663:NADPH Oxidase 4; D019255:NADPH Oxidases; C498910:NOX4 protein, human; C086986:CYBA protein, human; D009252:NADPH Dehydrogenase