N-nitroso-N-methylurea and N-nitroso-N-ethylurea induce upregulation of cellular NF-kappa B activity through protein kinase C-dependent pathway in human malignant keratinocytes

The upregulatory mechanism of cellular NF-kappaB activity by carcinogens, N-nitroso-N-methylurea (NMU) and N-nitroso-N-ethylurea (NEU) in human malignant keratinocytes was investigated. To elucidate the role of protein kinase C (PKC) in the upregulation of NF-kappaB by NMU and NEU, two known PKC inhibitors, staurosporine and H-7 were studied. Treatment of cells with PKC inhibitors decreased NF-kappaB activity in a dose responsive manner at concentrations of 20 approximately 200 nM. Staurosporine (160 nM) and H-7 (200 nM) downregulated the cellular NF-kappaB activation up to 20 and 60% compared to the NF-kappaB activity that was upregulated by NMU (5 microM) and NEU (5 microM), respectively. These results indicated that the PKC activity was responsible for the upregulation of NF-kappaB activity. The level of phosphorylation of I-kappaBalpha, the predominant form of the I-kappaB family represented by NMU and NEU, was quantified. The relative amount of I-kappaBalpha phosphorylation (serines-32 and -36) determined using the cellular activation of signaling ELISA assay method showed that NMU (5 microM) and NEU (5 microM) increased the amount of I-kappaBalpha phosphorylation up to 17 and 10% compared to the control, respectively. The results demonstrate the upregulatory effect of NMU and NEU on cellular NF-kappaB activity in human keratinocytes via the protein kinase C-mediated pathway.