EXPRESSION OF DOMINANT-NEGATIVE JUN INHIBITS ELEVATED AP-1 AND NF-KAPPA-B TRANSACTIVATION AND SUPPRESSES ANCHORAGE-INDEPENDENT GROWTH OF HPV IMMORTALIZED HUMAN KERATINOCYTES

AP-1 transactivation appears to be required for mouse JB6 cell neoplas tic transformation induced by the tumor promoter TPA or epidermal grow th factor (EGF). Exposure to AP-1 transrepressing retinoids and glucoc orticoids and expression of a dominant negative c-jun (TAM67) blocked tumor promoter-induced AP-1 transactivation and neoplastic transformat ion. The aim of the present study was to extend the inquiry of the rol e of AP-1 and other transcription factors to human neoplastic progress ion. Expression of human papilloma-virus (HPV) 16 or 18 E6 and E7 immo rtalizes human keratinocytes and inhibits serum/calcium-stimulated dif ferentiation. Further transformation by v-fos coexpression renders the se keratinocytes tumorigenic in nude mice. We have analysed two series of E6/E7 immortalized human keratinocyte cell lines that show progres sing phenotypes ranging from differentiation sensitive to anchorage-in dependent to tumorigenic in nude mice. We analysed the activities of A P-1 and NF-kappa B which may 'cross-talk'. Both DNA binding and transa ctivation of AP-1 and NF-kappa B transcription factors showed elevatio n in the anchorage-independent (16RH) and tumorigenic (18 v-fos) kerat inocyte lines compared to the less progressed but immortalized cell li nes. HPV E7 was expressed at a constant level shown by quantitative RT -PCR in both the more and the less progressed lines, indicating that E 7 is not the factor limiting this progression. Blocked shift/supershif t analysis indicates that Fos family member proteins especially Fra-1 and Fra-2 are related to progression and no changes found in the Jun f amily member proteins although they are present in the AP-1/DNA bindin g complex. When a dominant negative mutant c-jun driven by a human ker atin 14 promoter was cotransfected with AP-1 or NF-kappa B reporters, both AP-1 and NF-kappa B activities were suppressed in the more progre ssed cell lines 16RH add 18 v-fos but not in the less progressed 16RL or 18 cell lines. Overexpression of the same dominant negative c-jun d id not inhibit p53 dependent reporter transactivation, indicating the specificity of inhibition of AP-1 and NF-kappa B transactivation in th e HPV-immortalized cells. Stable transfectants of this mutant c-jun in the two more progressed cell lines 16RH and 18 v-fos showed reduced A P-1 and NF-kappa B activation and reduced anchorage-independent growth . Together, these results indicate that activation of AP-1, NF-kappa B or both may contribute to neoplastic progression in HPV immortalized human keratinocytes and that specific targeting of the elevated levels seen in benign or malignant tumors might be effective for prevention or treatment of human cancer.