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A significant decrease of the transcriptional activity of p53 mutants deriving from human functional adrenal tumors
Abstract:
Recently, our laboratory has found a high incidence (77%) of p53 gene mutations in human functional adrenal tumors. Furthermore, the majority of mutant sites were assembled at codons 100, 102, and 249. These mutation sites are not common, and there have been no studies addressing whether or not these mutants points or mutant styles cause the p53 protein to lose function. It has been well known that p53 is a transcription factor. To examine the transcriptional activities of these mutant p53 genes from patients with functional adrenal tumors, we constructed p53 expression plasmids from tumors and paired adjacent normal adrenal gland tissues, using a transient co-transfection assay with a reporter gene in H358 cells. Wild-type p53 from normal adrenal gland tissues specifically trans-activates the expression of a chloramphenicol acetyltransferase (CAT) reporter gene in H358 cells. Three mutant p53 proteins (at codons 100, 102, and 249, respectively) from tumors showed a >90% loss of transcriptional activity. One mutant at codon 68, other than at hot spots, remained at approximately 65% transcriptional activity. An immunoprecipitation assay showed that the mutant proteins of codon 68 and codon 102 could respond to the three monoclonal antibodies (PAbDO-1, PAb1620, and PAb421), indicating that there were no obvious changes in the antigenicity of the proteins. However, the mutant protein of codon 249 could not respond to the carboxy-terminus-specific antibody PAb421 and conformation-specific antibody PAb1620, indicating that there were some obvious changes in the conformation of the mutant proteins. The mutant protein of codon 100 could not be detected by immunoprecipitation assay but could be analyzed by Western blot. In a further study using a DNA-binding assay, it was shown that the loss of transcriptional activity was caused by the loss of DNA-binding ability. These results show that the p53 mutants, derived from functional adrenal tumors, actually lost DNA-binding ability and decreased the transcriptional activity. However, the role of the mutant protein in the tumorigenesis of functional adrenal tumors requires further investigation.
Insights
Mutations in the p53 gene are common in adrenal tumors. These p53 gene mutations lead to a loss of DNA-binding ability and decreased transcriptional activity, impacting tumor development.
Area of Science:
- Oncology
- Molecular Biology
- Genetics
Background:
- The p53 gene is a critical tumor suppressor.
- A high incidence (77%) of p53 gene mutations has been observed in human functional adrenal tumors.
- Specific mutation sites at codons 100, 102, and 249 are frequently found in these tumors.
Purpose of the Study:
- To investigate the functional consequences of p53 gene mutations found in functional adrenal tumors.
- To determine if these specific p53 mutants exhibit altered transcriptional activity.
- To assess the DNA-binding ability and protein conformation of mutant p53.
Main Methods:
- Construction of p53 expression plasmids from tumor and normal adrenal tissues.
- Transient co-transfection assay with a reporter gene (CAT) in H358 cells.
- Immunoprecipitation and Western blot assays to analyze mutant p53 protein expression and conformation.
- DNA-binding assays to evaluate the impact of mutations on DNA binding.
Main Results:
- Mutant p53 proteins at codons 100, 102, and 249 showed a >90% loss of transcriptional activity.
- Mutant p53 at codon 68 retained approximately 65% transcriptional activity.
- Loss of transcriptional activity correlated with impaired DNA-binding ability.
- Mutant p53 at codon 249 exhibited conformational changes, while codon 100 mutant was difficult to detect via immunoprecipitation.
Conclusions:
- p53 mutants found in functional adrenal tumors exhibit significantly reduced transcriptional activity due to loss of DNA-binding ability.
- Specific mutations at codons 100, 102, and 249 disrupt p53 function.
- Further research is needed to elucidate the role of these mutant p53 proteins in the tumorigenesis of functional adrenal tumors.