The host environment promotes the constitutive activation of nuclear factor-kappa B and proinflammatory cytokine expression during metastatic tumor progression of murine squamous cell carcinoma

We reported previously that tumor cells isolated from metastases of the in vitro transformed squamous cell carcinoma Line Pam 212 exhibit an elevation in constitutive production of proinflammatory cytokines interleukin (IL)-1 alpha, IL-6, granulocyte-macrophage colony-stimulating factor, and KC (the murine homologue of chemokine Gro-alpha). The basis for constitutive expre ssion of these cytokines after tumor progression in vivo is unknown. Regula tion of the expression of these proinflammatory cytokines involves transcri ption factor nuclear factor kappa B (NF-kappa B), which can be activated by cytokines such as tumor necrosis factor (TNF)-alpha. In this study, we com pared the constitutive and TNF-alpha-induced expression of proinflammatory cytokines in parental Pam 212 and metastatic LY-2 and LY-8 cell lines and d etermined the relationship of cytokine expression to activation of NF-kappa B, We found that the metastatic cell lines exhibited an increase in consti tutive and TNF-alpha-inducible expression of proinflammatory cytokines when compared with parental Pam 212 cells, The increased cytokine expression wa s associated with an increase in constitutive and TNF-alpha-inducible activ ation of NF-kappa B as demonstrated by electrophoretic mobility shift assay and luciferase-reporter gene assay. Constitutive nuclear localization of N F-kappa B p65 was observed in LY-2 and LY-8 cells in culture and in tumor s pecimens but rarely in Pam 212 cells, consistent with the constitutive acti vation of NF-kappa B in tumor cells after selection in vivo. Induction of N F-kappa B by TNF-alpha was inhibited by the addition of protease inhibitors calpain inhibitor I and N-tosyl-phechloromethyl ketone and antioxidant 1-p yrrolidinecarbodithioic acid, whereas constitutive activation of NF-kappa B and cytokine KC mRNA expression was inhibited by N-tosyl-phechloromethyl k etone alone. Overexpression of a human I kappa B alpha dominant suppresser in Pam 212 cells inhibited TNF-alpha-induced NF-kappa B binding activity an d KC expression. These data indicate that activation of NF-kappa B contribu tes to increased expression of proinflammatory cytokines during metastatic tumor progression of squamous cell carcinoma, and that distinct mechanisms may be involved in the regulation of constitutive and TNF-alpha-induced act ivation of NF-kappa B in squamous cell carcinoma.