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Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function
Annemieke de Vries1, Elsa R Flores, Barbara Miranda
1Department of Biology and Center for Cancer Research, and Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Abstract:
The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.
Insights
Mutant p53 protein can inhibit wild-type p53 function, impairing tumor suppression and cancer treatment response. These findings highlight the dominant-negative effects of common p53 mutations.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p53 tumor suppressor gene is frequently mutated in human cancers.
- p53 mutations, often missense, occur in the DNA-binding domain and can confer familial cancer predisposition.
- Mutant p53 is hypothesized to inhibit wild-type p53 function through a dominant-negative mechanism.
Purpose of the Study:
- To investigate the dominant-negative inhibition of wild-type p53 by two common point mutations (R270H and P275S).
- To analyze the effects of these mutant p53 proteins expressed at physiological levels in normal cells.
Main Methods:
- Targeted R270H and P275S mutations into the genome of mouse embryonic stem cells.
- Analysis of p53 downstream target gene transcriptional activation following gamma irradiation.
- Assessment of doxorubicin-induced apoptosis in mutant versus wild-type cells and thymocytes.
Main Results:
- Heterozygous point-mutated p53 alleles delayed transcriptional activation of p53 target genes after gamma irradiation.
- Doxorubicin-induced apoptosis was significantly impaired in mutant embryonic stem cells and thymocytes.
- p53-independent apoptotic pathways remained intact in heterozygous mutant cells.
Conclusions:
- Physiological expression of point-mutated p53 strongly limits cellular p53 function, confirming dominant-negative activity.
- Cells heterozygous for p53 mutations may exhibit compromised tumor suppression and reduced response to chemotherapy.