Induced expression of dominant-negative c-jun downregulates NFkappaB 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 (NFkappaB) transcription factors. The present study addresses the question of whether elevated NFkappaB 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 NFkappaB- 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/NFkappaB 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 NFkappaB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFkappaB p65, suggesting that inhibition of NFkappaB 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 NFkappaB 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 (NFkappaB responsive). These results indicate that genes controlled by NFkappaB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFkappaB activation through direct interaction with NFkappaB 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.
Li
J J
JJ
Gene Regulation Section, National Cancer Institute, National Institutes of Health, Frederick, Maryland 21702-1201, USA.
Cao
Y
Y
Young
M R
MR
Colburn
N H
NH
D002448:Cell Adhesion / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002461:Cell Line, Transformed; D002471:Cell Transformation, Neoplastic / Q000235:genetics*; D004247:DNA / Q000378:metabolism; D015536:Down-Regulation; D005786:Gene Expression Regulation; D016758:Genes, jun / Q000235:genetics*; D006801:Humans; D015603:Keratinocytes / Q000502:physiology*; D008156:Luciferases / Q000096:biosynthesis / Q000235:genetics; D016328:NF-kappa B / Q000037:antagonists & inhibitors / Q000235:genetics*; D010641:Phenotype; D016755:Proto-Oncogene Proteins c-jun / Q000096:biosynthesis / Q000235:genetics / Q000502:physiology*; D012097:Repressor Proteins / Q000235:genetics*; D018808:Transcription Factor AP-1 / Q000037:antagonists & inhibitors / Q000235:genetics*; D051996:Transcription Factor RelA; D015533:Transcriptional Activation; D014162:Transfection; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016328:NF-kappa B; D016755:Proto-Oncogene Proteins c-jun; D012097:Repressor Proteins; D018808:Transcription Factor AP-1; D051996:Transcription Factor RelA; D016159:Tumor Suppressor Protein p53; C034811:tetracycline resistance-encoding transposon repressor protein; D004247:DNA; D008156:Luciferases