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Inhibition of cyclooxygenase-2 gene expression by p53
K Subbaramaiah1, N Altorki, W J Chung
1Department of Medicine, The New York Presbyterian Hospital-Cornell, New York, New York 10021, USA.
Abstract:
Oncogenes enhance the expression of cyclooxygenase (Cox)-2, but interactions between tumor suppressor genes and Cox-2 have not been studied. In the present work, we have compared the levels of Cox-2 and the production of prostaglandin E2 in mouse embryo fibroblasts that do not express any p53 ((10)1) versus the same cell line ((10. 1)Val5) engineered to overexpress wild-type (wt) p53 at 32 degrees C or mutant p53 at 39 degrees C. Cells expressing wt p53 showed about a 10-fold decrease in synthesis of prostaglandin E2 compared with those expressing mutant p53. Levels of Cox-2 protein and mRNA were markedly suppressed by wt p53 but not by mutant p53. Nuclear run-offs revealed decreased rates of Cox-2 transcription in cells expressing wt p53. The activity of the Cox-2 promoter was reduced by 85% in cells expressing wt p53 but was reduced only by 30% in cells expressing mutant p53 compared with cells null for p53. The effect of p53 on the suppression of Cox-2 promoter activity was localized to the first 40 base pairs 5' from the transcription start site. Electrophoretic mobility shift assay revealed that p53 competed with TATA-binding protein for binding to mouse Cox-2 or human Cox-2 promoter extending from -50 to +52 base pairs. The results of this study suggest that interactions between p53 and Cox-2 could be important for understanding why levels of Cox-2 are undetectable in normal cells and increased in many tumors.
Insights
Wild-type p53 significantly suppresses cyclooxygenase-2 (Cox-2) expression and prostaglandin E2 production. This interaction, mediated by p53 binding to the Cox-2 promoter, may explain Cox-2 levels in normal and tumor cells.
Area of Science:
- Molecular Biology
- Cancer Research
- Biochemistry
Background:
- Oncogenes increase cyclooxygenase-2 (Cox-2) expression.
- The role of tumor suppressor genes, specifically p53, in regulating Cox-2 has not been investigated.
- Cox-2 and prostaglandin E2 are implicated in tumorigenesis.
Purpose of the Study:
- To investigate the interaction between p53 and Cox-2 expression.
- To determine how wild-type (wt) p53 and mutant p53 affect Cox-2 levels and prostaglandin E2 production.
Main Methods:
- Comparison of Cox-2 and prostaglandin E2 levels in p53-null versus p53-expressing mouse embryo fibroblasts.
- Analysis of Cox-2 mRNA and protein levels.
- Nuclear run-off assays to assess Cox-2 transcription rates.
- Reporter assays to measure Cox-2 promoter activity.
- Electrophoretic mobility shift assays (EMSA) to study p53 binding to the Cox-2 promoter.
Main Results:
- Wild-type p53 suppressed prostaglandin E2 synthesis by approximately 10-fold compared to mutant p53.
- Wild-type p53 markedly reduced Cox-2 protein and mRNA levels, while mutant p53 had minimal effect.
- Cox-2 transcription rates and promoter activity were significantly decreased by wt p53.
- p53 binding to the Cox-2 promoter was confirmed via EMSA, showing competition with TATA-binding protein.
Conclusions:
- Wild-type p53 actively suppresses Cox-2 expression and prostaglandin E2 production.
- The interaction between p53 and the Cox-2 promoter is crucial for regulating Cox-2 levels.
- These findings provide insights into the regulation of Cox-2 in normal tissues and its overexpression in various cancers.