AP-1 transactivation appears to be required for mouse JB6
cell neoplastic transformation induced by the
tumor promoter TPA or
epidermal growth factor (
EGF). Exposure to
AP-1 transrepressing
retinoids and
glucocorticoids and expression of a dominant negative c-jun (TAM67) blocked
tumor promoter-induced
AP-1 transactivation and neoplastic transformation. The aim of the present study was to extend the inquiry of the role of
AP-1 and other
transcription factors to human neoplastic progression. Expression of human papillomavirus (HPV) 16 or 18 E6 and E7 immortalizes human keratinocytes and inhibits serum/
calcium-stimulated differentiation. Further transformation by v-fos co-expression renders these keratinocytes tumorigenic in nude mice. We have analysed two series of E6/E7 immortalized human keratinocyte cell lines that show progressing phenotypes ranging from differentiation sensitive to anchorage-independent to tumorigenic in nude mice. We analysed the activities of
AP-1 and
NF-kappaB which may 'cross-talk'. Both
DNA binding and transactivation of
AP-1 and
NF-kappaB transcription factors showed elevation in the anchorage-independent (16RH) and tumorigenic (18 v-fos) keratinocyte lines compared to the less progressed but immortalized cell lines. HPV E7 was expressed at a constant level shown by quantitative RT-PCR in both the more and the less progressed lines, indicating that E7 is not the factor limiting this progression. Blocked shift/supershift analysis indicates that Fos family member
proteins especially Fra-1 and Fra-2 are related to progression and no changes found in the Jun family member
proteins although they are present in the AP-1/
DNA binding complex. When a dominant negative mutant c-jun driven by a human
keratin 14 promoter was co-transfected with
AP-1 or
NF-kappaB reporters, both
AP-1 and
NF-kappaB activities were suppressed in the more progressed cell lines 16RH and 18 v-fos but not in the less progressed 16RL or 18 cell lines. Overexpression of the same dominant negative c-jun did not inhibit p53 dependent reporter transactivation, indicating the specificity of inhibition of
AP-1 and
NF-kappaB transactivation in the HPV-immortalized cells. Stable transfectants of this mutant c-jun in the two more progressed cell lines 16RH and 18 v-fos showed reduced
AP-1 and
NF-kappaB activation and reduced anchorage-independent growth. Together, these results indicate that activation of
AP-1,
NF-kappaB 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.