Constitutive activation of transcription factors NF-kappa B, AP-1, and NF-IL6 in human head and neck squamous cell carcinoma cell lines that express pro-inflammatory and pro-angiogenic cytokines

We previously reported that human head and neck squamous cell carcinomas (H NSCCs) express the proinflammatory and pro-angiogenic cytokines interleukin (IL)-1 alpha, IL-6, IL-8, and granulocyte-macrophage colony stimulating fa ctor in vitro and in vive. The promoter region of the genes encoding these cytokines include binding sites for the transcription factors nuclear facto r (NF) KB/Rel A, activator protein-1 (AP-1), and CCAAT enhancer-binding pro tein beta (C/EBPP, or NF-IL6), which have been reported to contribute to ac tivation of these cytokine genes. In the study presented here, we examined the activation, composition, and function of these transcription factors in HNSCC cell lines that express pro-inflammatory cytokines, by using electro phoretic mobility shift and reporter-gene assays. Constitutive activation o f NF-kappa B, AP-1, and NF-IL6 DNA-binding proteins was detected. Supershif t analysis with antibodies specific for NF-kappa B, AP-1, and NF-IL6 bindin g proteins showed that the NF-kappa B-binding protein included p65/Rel A an d p50; AP-1 activity included c-jun, junB, junD, and Fra-1; and NF-IL6 incl uded C/EBPP. Mutational analysis of the NF-kappa B, AP-1, and NF-IL6 sites in the IL-8 promoter region showed that NF-kappa B and AP-1 sites contribut ed to constitutive IL-8 reporter activity in HNSCC. HNSCC lines that exhibi ted increased IL-8 secretion relative to simian virus 40-immortalized and p rimary keratinocyte cell lines also demonstrated a concordant increase in N F-kappa B reporter activity relative to nonmalignant keratinocytes. We conc luded that the early transcription factors NF-kappa B, AP-1, and NF-IL6 are constitutively activated in human HNSCC cell lines and that NF-kappa B and AP-1 promote expression of the proinflammatory and pro-angiogenic cytokine IL-8 in HNSCC. The demonstration of the activation of these transcription factors will be helpful in defining the identity and role of these and othe r early gene products that contribute to pathogenesis of the malignant phen otype in HNSCC and in defining potential targets for pharmacologic and mole cular therapy of HNSCC. Published 1999 Wiley-Liss, Inc.(dagger).