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The mitogen activated protein (MAP) kinases or extracellular signal-regulated kinases (Erks) are activated in response to Ras expression or exposure to tumor promoters or to growth factors, and have been implicated in AP-1 transactivation in some models. We have shown that tumor promoter induced activation of the transcription factor AP-1 is required for induced neoplastic transformation in the Balb/C JB6 cell model. Jun and Fos family protein levels have been found not to be limiting for AP-1 response. The present study asks whether activation of Erks1 and 2 is required for AP-1 transactivation and transformation of JB6 cells and whether Erks might be targeted for cancer prevention. Expression of either of two different dominant negative kinase inactive Erk2 mutants in transformation sensitive (P+) JB6 cells substantially inhibited the tumor promoter induced activation of Erks1 and 2 and of AP-1 measured by a collagenase-luciferase reporter. Multiple mutant Erk2 expressing clonal lines were also rendered non-responsive to induced neoplastic transformation. These observations, together with our recent finding attributing AP-1 non-responsiveness to Erk deficiency in a clonal line of transformation resistant (P-) cells, argue for a requirement for Erks1 and/or 2 activation in AP-1 transactivation in the mouse JB6 neoplastic progression model, and suggest the utility of Erks as a prevention target.

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.

Human manganese containing superoxide dismutase (MnSOD) cDNA was transfected into a human breast cancer cell line (MCF-7) in order to examine the effect of increased functional MnSOD on the cellular phenotype. A MnSOD-overexpressing clone was compared to control vector-transfected cells and to wild type MCF-7 cells. Southern blotting indicated incorporation of MnSOD cDNA into genomic DNA in the MnSOD overexpressing cell line. The MnSOD overexpressing cell line showed a 5.7-fold increase in MnSOD activity compared to wild type MCF-7 cells. Similar increases in MnSOD immunoreactive protein and mRNA levels were observed by Western and Northern blotting as well as using RT-PCR. The plating efficiency of cells grown in different concentrations of serum (1 to 20%) was decreased in the MnSOD overexpressing cell line. The clonogenic fraction in soft agar culture was also decreased after MnSOD cDNA transfection. When inoculated in nude mice, tumor growth was markedly inhibited in MnSOD overexpressing cells compared to wild type MCF-7 cells or plasmid control cells. These results support the hypothesis that increased MnSOD expression suppresses the malignant phenotype of human breast cancer cells and suggests that the MnSOD gene is a tumor suppressor gene in human breast cancer.