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Activating mutations in p53 produce a common conformational effect. A monoclonal antibody specific for the mutant
J V Gannon1, R Greaves, R Iggo
1Molecular Immunochemistry Laboratory, Imperial Cancer Research Fund, Potters Bar, Herts, UK.
Abstract:
Point mutations in the p53 gene are the most frequently identified genetic change in human cancer. They convert murine p53 from a tumour suppressor gene into a dominant transforming oncogene able to immortalize primary cells and bring about full transformation in combination with an activated ras gene. In both the human and murine systems the mutations lie in regions of p53 conserved from man to Xenopus. We have developed a monoclonal antibody to p53 designated PAb240 which does not immunoprecipitate wild type p53. A series of different p53 mutants all react more strongly with PAb240 than with PAb246. The PAb240 reactive form of p53 cannot bind to SV40 large T antigen but does bind to HSP70. In contrast, the PAb246 form binds to T antigen but not to HSP70. PAb240 recognizes all forms of p53 when they are denatured. It reacts with all mammalian p53 and chicken p53 in immunoblots. We propose that immunoprecipitation of p53 by PAb240 is diagnostic of mutation in both murine and human systems and suggest that the different point mutations which convert p53 from a recessive to a dominant oncogene exert a common conformational effect on the protein. This conformational change abolishes T antigen binding and promotes self-oligomerization. These results are consistent with a dominant negative model where mutant p53 protein binds to and neutralizes the activity of p53 in the wild type conformation.
Insights
Point mutations in the p53 gene are common in human cancers, converting p53 into a dominant oncogene. A new antibody, PAb240, specifically detects these mutant p53 forms, aiding cancer diagnosis.
Area of Science:
- Molecular Biology
- Oncology
- Genetics
Background:
- Point mutations in the p53 gene are frequent in human cancers.
- Mutant p53 acts as a dominant oncogene, promoting cell immortalization and transformation.
Purpose of the Study:
- To develop a diagnostic tool for mutant p53.
- To investigate the conformational changes associated with p53 mutations.
Main Methods:
- Development of a monoclonal antibody, PAb240, specific for mutant p53.
- Immunoprecipitation and immunoblotting assays using PAb240 and PAb246 antibodies.
- Analysis of p53 binding to SV40 large T antigen and HSP70.
Main Results:
- PAb240 specifically immunoprecipitates mutant p53, not wild-type p53.
- Mutant p53 recognized by PAb240 shows altered binding properties, including reduced T antigen binding and increased HSP70 binding.
- PAb240 detects all denatured forms of p53, reacting with mammalian and chicken p53.
Conclusions:
- Immunoprecipitation with PAb240 is a diagnostic marker for p53 mutations in human and murine systems.
- p53 mutations induce a common conformational change, abolishing T antigen binding and promoting self-oligomerization.
- Mutant p53 may function via a dominant-negative mechanism, neutralizing wild-type p53 activity.