Expression of dominant negative Jun inhibits elevated AP-1 and NF-kappaB transactivation and suppresses anchorage independent growth of HPV immortalized human keratinocytes.
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.
Li
J J
JJ
Laboratory of Biochemical Physiology, National Cancer Institute, FCRDC, Frederick, Maryland 21702-1201, USA.
Rhim
J S
JS
Schlegel
R
R
Vousden
K H
KH
Colburn
N H
NH
D002455:Cell Division; D002460:Cell Line; D002472:Cell Transformation, Viral; D004268:DNA-Binding Proteins / Q000378:metabolism; D051921:Fos-Related Antigen-2; D006801:Humans; D053547:Keratin-14; D015603:Keratinocytes / Q000166:cytology* / Q000378:metabolism; D007633:Keratins / Q000235:genetics; D016296:Mutagenesis; D016328:NF-kappa B / Q000235:genetics*; D016761:Oncogene Proteins v-fos / Q000378:metabolism; D009856:Oncogene Proteins, Viral / Q000235:genetics; D027383:Papillomaviridae / Q000235:genetics*; D050725:Papillomavirus E7 Proteins; D010641:Phenotype; D011401:Promoter Regions, Genetic; D016760:Proto-Oncogene Proteins c-fos / Q000378:metabolism; D016755:Proto-Oncogene Proteins c-jun / Q000235:genetics / Q000378:metabolism*; D012097:Repressor Proteins; D018808:Transcription Factor AP-1 / Q000235:genetics*; D014157:Transcription Factors / Q000378:metabolism; D015533:Transcriptional Activation; D014162:Transfection; D000099169:Fos-Related Antigen 1
D004268:DNA-Binding Proteins; D051921:Fos-Related Antigen-2; D053547:Keratin-14; D007633:Keratins; D016328:NF-kappa B; D016761:Oncogene Proteins v-fos; D009856:Oncogene Proteins, Viral; D050725:Papillomavirus E7 Proteins; D016760:Proto-Oncogene Proteins c-fos; D016755:Proto-Oncogene Proteins c-jun; D012097:Repressor Proteins; D018808:Transcription Factor AP-1; D014157:Transcription Factors; D000099169:Fos-Related Antigen 1; C068651:E6 protein, Human papillomavirus type 16; C052603:E6 protein, Human papillomavirus type 18; C052633:E7 protein, Human papillomavirus type 18; C497872:FOSL2 protein, human; C507540:KRT14 protein, human; C507446:Krt14 protein, mouse; C059731:oncogene protein E7, Human papillomavirus type 16