Expression of dominant negative Jun inhibits elevated AP-1 and NF-κB transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes

Abstract

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 dierentiation. Further transformation by v-fos coexpression 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 dierentiation sensitive to anchorage-independent to tumorigenic in nude mice. We analysed the activities of AP-1 and NFkB which may`cross-talk'. Both DNA binding and transactivation of AP-1 and NF-kB 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 cjun driven by a human keratin 14 promoter was cotransfected with AP-1 or NF-kB reporters, both AP-1 and NF-kB 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 speci®city of inhibition of AP-1 and NF-kB 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-kB activation and reduced anchorage-independent growth. Together, these results indicate that activation of AP-1, NF-kB or both may contribute to neoplastic progression in HPV immortalized human keratinocytes and that speci®c targeting of the elevated levels seen in benign or malignant tumors might be eective for prevention or treatment of human cancer.

Tumor promoter-induced ornithine decarboxylase gene expression occurs independently of AP-1 activation

“…The dominant negative c-Jun (TAM67) used in these experiments has also been shown to trans-repress NFkB-dependent transactivation (Dong et al, 1997;Li et al, 1998). An HIV-NF-kB collagenase reporter construct was used to show inhibited NF-kB-dependent transcription when co-transfected with TAM67 in mouse keratinocytes or HPV immortalized human keratinocytes (Dong et al, 1997;Li et al, 1998).…”

“…An HIV-NF-kB collagenase reporter construct was used to show inhibited NF-kB-dependent transcription when co-transfected with TAM67 in mouse keratinocytes or HPV immortalized human keratinocytes (Dong et al, 1997;Li et al, 1998). The NF-kB/Rel transcription factor family has been shown to regulate c-Myc gene transcription in immature B lymphocytes (Lee et al, 1995).…”

“…There is also evidence that AP-1 induction is not necessary for proliferative stimulus or hyperplasia. JB6 P 7 transformation-insensitive cells are still mitogenically responsive, and TAM67 expression in other cell lines also did not inhibit growth yet blocked transformation (Dong et al, 1994(Dong et al, , 1997Li et al, 1998). Transgenic mice overexpressing TAM67 in the skin show AP-1 inhibition, but hyperplasia still occurs following TPA treatments (Young et al, submitted).…”

“…From studies utilizing reporter constructs measuring AP-1-dependent gene expression, the induction of AP-1 transactivation also has been shown to be essential for tumor promotion and progression in cell culture models (Dong et al, 1994(Dong et al, , 1997Li et al, 1998;Domann et al, 1994). In JB6 and human keratinocyte models, expression of dominant negative c-jun can block induced AP-1 activity and neoplastic transformation (Dong et al, 1994(Dong et al, , 1997Li et al, 1998;Domann et al, 1994).…”

“…In JB6 and human keratinocyte models, expression of dominant negative c-jun can block induced AP-1 activity and neoplastic transformation (Dong et al, 1994(Dong et al, , 1997Li et al, 1998;Domann et al, 1994). TPA-induced upregulation of the AP-1 transcription factor complex occurs prior to TPAinduced ODC gene expression.…”