AP-1, NF kappa B, and ERK activation thresholds for promotion of neoplastic transformation in the mouse epidermal JB6 model

The promotion-sensitive mouse epidermal JB6 cells (done 41) have been used to identify the tumor-promoting activity of various compounds. Because treatment by tumor promoters [12-O- tetradecanoylphorbol-13-acetate (TPA), epidermal growth factor (EGF), or tumor necrosis factor alpha (TNFalpha)] transforms done 41 cells to anchorage-independent and tumorigenic phenotypes, they are considered to be undergoing late-stage tumor promotion. Here we address the question of how much activation of transformation-relevant transcription factors [activator protein-1 (AP-1), ternary complex factors (TCFs), or nuclear factor kappaB (NFkappaB)] is required for transformation response and how much tumor promoter produces significant risk of transformation. Stable transfectants harboring a reporter construct with an AP-1 response element, serum-response element (SRE), or NFkappaB response element were established. We examined the relationship between concentration of tumor promoters, key signaling events, and activation of the transcription factors. A concentration of >0.2 nM TPA or 0.12 ng/mL (0.02 nM) EGF produced a significant increase in transformation response as well as in extracellular signal- regulated protein kinase (ERK), SRE, or AP-1 activation. Treatment with >0.4 U/mL (2.35 pM) TNFalpha increased NFkappaB activity and transformation response in a dose-dependent manner. However, transformation response decreased at >33 U/mL TNFalpha due to a cytotoxic response. These findings suggest that the signaling pathway leading to the activation of ERK, TCF, and AP-1 proteins constitutes a major factor determining the risk of tumor promotion by TPA or EGF. Cell toxicity in addition to NFkappaB activation should be considered in predicting TNFalpha-induced transformation response.

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