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

Jian-Jian Li, Ya Cao, Mathew R. Young, Nancy H. Colburn

Research output: Contribution to journalArticle

71 Citations (Scopus)

Abstract

Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor κB (NFκB) transcription factors. The present study addresses the question of whether elevated NFκB 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 NFκB- 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/NFκB 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 NFκB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFκB p65, suggesting that inhibition of NFκB 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 NFκB 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 (NFκB responsive). These results indicate that genes controlled by NFκB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFκB activation through direct interaction with NFκB 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.

Original languageEnglish (US)
Pages (from-to)159-169
Number of pages11
JournalMolecular Carcinogenesis
Volume29
Issue number3
DOIs
StatePublished - 2000
Externally publishedYes

Fingerprint

Transcription Factor AP-1
Keratinocytes
Down-Regulation
Phenotype
Genes
Neoplasms
Transcriptional Activation
CCAAT-Enhancer-Binding Protein-beta
Gene Expression
Matrix Metalloproteinase 3
DNA
Collagenases
Tetracycline
Reverse Transcriptase Polymerase Chain Reaction
Interleukin-1
Immunoprecipitation
Genetic Therapy
Proteins
Transcription Factors
Carcinoma

Keywords

  • Activator protein 1
  • Human keratinocytes
  • Inducible transgene
  • Neoplastic progression
  • Nuclear factor κB

ASJC Scopus subject areas

  • Cancer Research
  • Molecular Biology

Cite this

Induced expression of dominant-negative c-jun downregulates NFκB and AP-1 target genes and suppresses tumor phenotype in human keratinocytes. / Li, Jian-Jian; Cao, Ya; Young, Mathew R.; Colburn, Nancy H.

In: Molecular Carcinogenesis, Vol. 29, No. 3, 2000, p. 159-169.

Research output: Contribution to journalArticle

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abstract = "Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor κB (NFκB) transcription factors. The present study addresses the question of whether elevated NFκB 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 NFκB- 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/NFκB 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 NFκB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFκB p65, suggesting that inhibition of NFκB 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 NFκB 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 (NFκB responsive). These results indicate that genes controlled by NFκB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFκB activation through direct interaction with NFκB 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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AB - Neoplastically transformed mouse and human keratinocytes elevate transactivation of both activator protein 1 (AP-1) and nuclear factor κB (NFκB) transcription factors. The present study addresses the question of whether elevated NFκB 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 NFκB- 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/NFκB 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 NFκB DNA-binding activity. Immunoprecipitation showed that TAM67 interacted in keratinocyte nuclei with NFκB p65, suggesting that inhibition of NFκB 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 NFκB 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 (NFκB responsive). These results indicate that genes controlled by NFκB and by AP-1 may be transformation-relevant targets of TAM67 and that TAM67 may inhibit NFκB activation through direct interaction with NFκB 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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