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Inhibition of DNA topoisomerase II alpha gene expression by the p53 tumor suppressor
Q Wang1, G P Zambetti, D P Suttle
1Department of Pharmacology, College of Medicine, University of Tennessee, Memphis 38163, USA.
Abstract:
DNA topoisomerase II (topo II) is an essential nuclear enzyme involved in major cellular functions such as DNA replication, transcription, recombination, and mitosis. While an elevated level of topo II alpha is associated with cell proliferation, wild-type (wt) p53 inhibits the expression of various growth-stimulatory genes. To determine if p53 downregulates topo II alpha gene expression, a murine cell line, (10)1val, that expresses a temperature-sensitive p53 was utilized. The (10)1val cells had significantly lower levels of topo II alpha mRNA and protein following incubation for 24 h at 32 degrees C (p53 with wt conformation) than at 39 degrees C (p53 with mutant conformation). The effect of p53 on the human topo II alpha gene promoter was determined by using luciferase reporter plasmids containing varying lengths of the topo II alpha promoter transiently cotransfected into p53-deficient (10)1 cells together with wt or mutant p53 expression plasmids. Transcription from the full-length (bp -557 to +90) topo II alpha promoter was decreased 15-fold by wt p53 in a concentration-dependent manner, whereas mutant p53 exerted much weaker inhibition. Consecutive deletion of the five inverted CCAAT elements (ICEs) from the topo II alpha promoter reduced both the basal promoter activity and wt p53-induced suppression. Transcription of the minimal promoter (-32 to +90), which contains no ICE, was slightly stimulated by wt or mutant p53 expression. When point mutations were introduced into the most proximal ICE (-68), the inhibitory effect of wt p53 was alleviated and stimulation of topo II alpha expression resulted. Our study suggests that wt p53 functions as a transcriptional repressor of topo II alpha gene expression, possibly through a functional interaction with specific ICEs. Inactivation of wt p53 may reduce normal regulatory suppression of topo II alpha and contribute to abortive cell cycle checkpoints, accelerated cell proliferation, and alterations in genomic stability associated with neoplasia.
Insights
Wild-type p53 (wt p53) suppresses DNA topoisomerase II alpha (topo II alpha) gene expression by interacting with inverted CCAAT elements (ICEs) in the promoter. This repression is crucial for regulating cell proliferation and genomic stability.
Area of Science:
- Molecular Biology
- Cell Biology
- Genetics
Background:
- DNA topoisomerase II (topo II) is vital for DNA replication, transcription, recombination, and mitosis.
- Elevated topo II alpha levels correlate with cell proliferation, while wild-type p53 (wt p53) typically inhibits growth-stimulatory genes.
Purpose of the Study:
- To investigate whether wt p53 downregulates topo II alpha gene expression.
- To elucidate the mechanism by which p53 influences topo II alpha transcription.
Main Methods:
- Utilized a murine cell line (101val) with temperature-sensitive p53 to assess topo II alpha mRNA and protein levels.
- Employed luciferase reporter assays with varying topo II alpha promoter lengths in p53-deficient cells, co-transfected with wt or mutant p53 expression plasmids.
Main Results:
- Wt p53 significantly reduced topo II alpha mRNA and protein levels.
- Wt p53 inhibited transcription from the full-length topo II alpha promoter by 15-fold in a dose-dependent manner.
- Mutations in inverted CCAAT elements (ICEs) of the promoter alleviated p53-mediated repression, suggesting ICEs are key interaction sites.
Conclusions:
- Wt p53 acts as a transcriptional repressor of the topo II alpha gene, likely via interaction with specific ICEs.
- Inactivation of wt p53 may lead to increased topo II alpha expression, contributing to uncontrolled cell proliferation and genomic instability in neoplasia.