CONSTITUTIVE AP-1 DNA-BINDING AND TRANSACTIVATING ABILITY OF MALIGNANT BUT NOT BENIGN MOUSE EPIDERMAL-CELLS

The mouse epidermal cell line 308 contains an activated Ha-ras gene an d forms benign papillomas when transplanted to the skin of athymic nud e mice. A radiation-associated malignant variant of this cell line, 30 8-10Gy5, has been isolated and shown to form squamous cell carcinomas in nude mice. To further examine the molecular events involved in mali gnant conversion of 308-10Cy5, we assessed the activator protein-1 (AP -1) binding and transactivating ability of 308 and 308-10Gy5. In nucle ar protein extracts of 308, AP-1 sequence-specific binding to an oligo nucleotide containing a single high-affinity AP-1 binding site was ind uced by the tumor promoter 12-O-tetradecanoylphorbol-13-acetate, as de termined by gel shift analysis. Nuclear extracts of 308-10Gy5 bound to the AP-1 oligonucleotide without treatment with tumor promoters. Not only was sequence-specific AP-1 DNA binding constitutively active in m alignant versus benign tumor cells, but so was transactivation of a un ique AP-1-responsive chloramphenicol acetyltransferase reporter constr uct, pTiCTaK. Constitutive transactivation of this AP-1-responsive rep orter construct was observed in the malignant but not the benign tumor cells. Furthermore, steady-state transcript levels of the tumor-assoc iated AP-1-responsive genes stromelysin, urokinase-type plasminogen ac tivator, c-jun, and c-fos were higher in malignant 308-10Gy5 cells tha n in benign 308 cells. These results suggest that acquisition of const itutive AP-1 DNA binding and transactivation can result in sustained d eregulation of gene expression. While malignant progression in keratin ocytes is probably not due solely to the acquisition of constitutive c ellular AP-1 activity, the effect of deregulated expression of AP-1-re gulated genes, especially basement membrane-degrading enzymes, may be functionally related to malignant conversion. (C) 1994 Wiley-Liss, Inc .