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Stable expression of a c-JUN deletion mutant in two malignant mouse epidermal cell lines blocks tumor formation in
F E Domann1, J P Levy, M J Birrer
1Department of Radiation Oncology, University of Arizona, Tucson 85724.
Abstract:
We have stably expressed a trans-activation suppressing deletion mutant of the human c-jun gene (TAM-67) in the malignant mouse epidermal cell lines 10Gy5 and PDV. Expression of the p26 mJUN protein blocked both constitutive and inducible transcriptional trans-activation of several AP-1 responsive reporter chloramphenicol acetyltransferase constructs. p26 mJUN was able to block both 12-O-tetradecanoylphorbol-13-acetate (TPA) and okadaic acid induced expression of the mouse stromelysin gene in 10Gy5 cells and TPA induced expression of the urokinase-type plasminogen activator gene in PDV cells as determined by Northern analyses. Both genes contain TPA response elements in their promoter regions and are known to be AP-1 responsive. The presence of p26 mJUN in nuclear extracts, as determined by Western blotting, did not detectably alter the DNA binding activity of endogenous AP-1 as determined by gel shift analysis with an oligonucleotide containing a single high affinity AP-1 binding site. UV cross-linking studies coupled with Western analyses identified DNA bound cJUN but not mJUN in nuclear extracts of stably transfected cell lines, suggesting that the mutant JUN protein may exert some of its antioncogenic effects in malignant mouse epidermal cells by a mechanism(s) not involving DNA binding. Malignant mouse epidermal cells which stably expressed the mutant JUN protein were not only inhibited in their AP-1 trans-activation response, but also in their ability to form s.c. tumors in nude mice. These results indicate that inhibition of AP-1 mediated transcriptional trans-activation alone can be sufficient to suppress the tumorigenic phenotype in a subset of malignant mouse epidermal cells.
Insights
A mutant human c-jun gene (TAM-67) suppressed tumor formation in mouse cells by blocking AP-1 transcriptional activity. This suggests inhibiting AP-1 trans-activation can suppress the tumorigenic phenotype.
Area of Science:
- Molecular Biology
- Cancer Research
- Gene Regulation
Background:
- Activating Protein-1 (AP-1) is a transcription factor complex involved in cell proliferation and differentiation.
- Dysregulation of AP-1 activity is implicated in the development of various cancers, including epidermal malignancies.
- The c-jun gene encodes a component of the AP-1 complex, and its altered expression can impact cellular behavior.
Purpose of the Study:
- To investigate the role of a trans-activation suppressing mutant of human c-jun (TAM-67) in malignant mouse epidermal cells.
- To determine if inhibiting AP-1 transcriptional activity can suppress the tumorigenic phenotype of these cells.
Main Methods:
- Stable expression of TAM-67 in 10Gy5 and PDV mouse epidermal cell lines.
- Reporter gene assays to assess AP-1 trans-activation.
- Northern blot analysis to measure gene expression of stromelysin and urokinase-type plasminogen activator.
- Western blot and gel shift analysis to evaluate AP-1 DNA binding activity.
Main Results:
- Expression of the p26 mJUN mutant protein inhibited both constitutive and inducible AP-1 trans-activation.
- AP-1 responsive gene expression, including stromelysin and urokinase-type plasminogen activator, was blocked by p26 mJUN.
- The mutant JUN protein did not alter endogenous AP-1 DNA binding but was not detected in DNA-bound complexes, suggesting a non-DNA binding mechanism of action.
- Malignant cells expressing p26 mJUN showed inhibited AP-1 trans-activation and reduced tumor formation in nude mice.
Conclusions:
- Inhibition of AP-1 mediated transcriptional trans-activation by the TAM-67 mutant is sufficient to suppress the tumorigenic phenotype in a subset of malignant mouse epidermal cells.
- The antioncogenic effects of the mutant JUN protein may involve mechanisms independent of direct DNA binding.
- Targeting AP-1 trans-activation represents a potential therapeutic strategy for epidermal cancers.