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Analysis of p53 mutants for transcriptional activity
L Raycroft1, J R Schmidt, K Yoas
1Department of Molecular Genetics, University of Texas M.D. Anderson Cancer Center, Houston 77030.
Abstract:
The wild-type p53 protein functions to suppress transformation, but numerous mutant p53 proteins are transformation competent. To examine the role of p53 as a transcription factor, we made fusion proteins containing human or mouse p53 sequences fused to the DNA binding domain of a known transcription factor, GAL4. Human and mouse wild-type p53/GAL4 specifically transactivated expression of a chloramphenicol acetyltransferase reporter in HeLa, CHO, and NIH 3T3 cells. Several mutant p53 proteins, including a mouse p53 mutant which is temperature sensitive for suppression, were also analyzed. A p53/GAL4 fusion protein with this mutation was also transcriptionally active only at the permissive temperature. Another mutant p53/GAL4 fusion protein analyzed mimics the mutation inherited in Li-Fraumeni patients. This fusion protein was as active as wild-type p53/GAL4 in our assay. Two human p53 mutants that arose from alterations of the p53 gene in colorectal carcinomas were 30- to 40-fold less effective at activating transcription than wild-type p53/GAL4 fusion proteins. Thus, functional wild-type p53/GAL4 fusion proteins activate transcription, while several transformation competent mutants do so poorly or not at all. Only one mutant p53/GAL4 fusion protein remained transcriptionally active.
Insights
Wild-type p53 protein suppresses cell transformation, while many mutant forms promote it. This study shows wild-type p53/GAL4 fusion proteins activate transcription, but most transformation-competent mutants do not.
Area of Science:
- Molecular Biology
- Cancer Research
- Genetics
Background:
- The p53 protein is a critical tumor suppressor.
- Mutant p53 proteins can promote cancer development.
- Understanding p53's transcriptional activity is key to cancer research.
Purpose of the Study:
- To investigate the role of p53 as a transcription factor.
- To compare the transcriptional activity of wild-type and mutant p53 proteins.
- To analyze p53 mutations associated with Li-Fraumeni syndrome and colorectal cancer.
Main Methods:
- Creation of p53/GAL4 fusion proteins using human and mouse p53 sequences.
- Transactivation assays using a chloramphenicol acetyltransferase reporter gene.
- Testing fusion protein activity in HeLa, CHO, and NIH 3T3 cell lines.
Main Results:
- Wild-type p53/GAL4 fusion proteins demonstrated specific transactivation.
- A temperature-sensitive p53 mutant fusion protein was active only at the permissive temperature.
- Mutant p53/GAL4 fusion proteins from colorectal carcinomas showed significantly reduced transcriptional activity.
- A Li-Fraumeni patient-associated mutant p53/GAL4 fusion protein retained high activity.
Conclusions:
- Functional wild-type p53/GAL4 fusion proteins are transcriptionally active.
- Many transformation-competent p53 mutants exhibit poor or no transcriptional activity.
- Specific p53 mutations may impair tumor suppressor functions by reducing transcriptional activity.