Induced expression of dominant-negative c-jun downregulates NF kappa B and AP-1 target genes and suppresses tumor phenotype in human keratinocytes

Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor kappaB (NF kappaB) transcription factors. The present study addresses the question of whether elevated NF kappaB in addition to elevated AP-1-dependent gene expression is necessary for maintaining the tumor cell phenotype. When a tetracycline-regulatable dominant-negative c-jun (TAM67, having a truncated transactivation domain) was expressed in tumorigenic human keratinocytes, AP-1- and NF kappaB- but not p53-dependent reporter activity was inhibited by 40-60%. Tumor phenotype, as measured by anchorage-independent growth, was inhibited by 90%. Neither AP-1/NF kappaB activation nor expression of tumor phenotype was inhibited in TAM67-harboring keratinocytes under noninducing conditions. Electrophoretic mobility shift analysis showed that induction of TAM67 expression slightly increased AP-1- but reduced NF kappaB DNA- binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NF kappaB p65, suggesting that inhibition of NF kappaB by TAM67 is mediated by direct protein-protein interactions, possibly producing decreased binding to DNA or inactivating p65. To analyze the putative effector genes that may be targeted by TAM67, expression of genes responsive to AP-1 or NF kappaB was measured by reverse transcriptase-polymerase chain reaction in TAM67 transfectants with or without TAM67 induction. Induction of TAM67 inhibited or reduced the expression of collagenase I, stromelysin I (AP-1 responsive), and interleukins 1 and 6 (NF kappaB responsive). These results indicate that genes controlled by NF kappaB and by AP-1 may be transformation- relevant targets of TAM67 and that TAM67 may inhibit NF kappaB activation through direct interaction with NF kappaB p65. Moreover, the findings provide proof for the principle of using inducible TAM67 as a gene therapy to suppress tumor phenotype in human carcinoma cells. Published 2000 Wiley-Liss. Inc.

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