Activation of nuclear factor-kappa B in human metastatic melanoma cells and the effect of oxidative stress

The biological basis for the general pharmacological resistance of human me lanoma is unknown. A unique biochemical feature of the melanocyte is the sy nthesis of melanin, which leads to the generation of hydrogen peroxide and the consumption of reduced glutathione, This activity produces a state of c hronic oxidative stress in these cells. We demonstrated previously that the expression of the c-jun family was dysregulated in metastatic melanoma cel ls compared,vith normal human melanocytes (D. T. Yamanishi et al., J. Inves t. Dermatol,, 97: 349-353, 1991), In the current investigation, we measured the levels of two major redox response transcription factors, nuclear fact or-KB (NF-KB) and activator protein-1, in metastatic melanoma cells and nor mal melanocytes and their response to oxidative stress, The basal DNA-bindi ng activity of NF-KB as measured by the electrophoretic mobility shift assa y in metastatic melanoma cells was increased 4-fold compared with that of n ormal melanocytes. This level of binding was paralleled by a 1.5- to 4-fold increase in the expression of p50 (NF-kappa B1), p65 (Rel-A), and I kappa B-alpha as measured by Northern blot analysis. In contrast, the expression of p75 (c-rel) was markedly decreased (60%) in melanoma cells compared with normal melanocytes. Following oxidative stress produced by enzyme-generate d H2O2, free H2O2, or incubation with buthionine sulfoximine, NF-kappa B bi nding activity increased 1.5- to 2.5-fold in melanoma cells (buthionine sul foximine > H2O2), but only slightly in normal melanocytes. In contrast, act ivator protein-1 binding activity was unaffected or increased in normal mel anocytes in response to oxidative stress, but was either unaffected or decr eased in melanoma cells. These results suggest that the redox regulation of melanoma cells at the molecular level is fundamentally different from norm al melanocytes and may offer a unique avenue for preventive or therapeutic intervention as well as new insights into the pathogenesis of melanocyte tr ansformation.