Characterization of the p53 mutants ability to inhibit p73 beta transactivation using a yeast-based functional assay.

Abstract

p53 is the most frequently altered tumor suppressor gene in a wide spectrum of human tumors. The large majority of p53 mutations observed in tumors are missense mutations. The p73 gene, encoding a protein with significant sequence similarity to p53, expresses multiple transcription-competent spliced variants, or transcription-incompetent forms (i.e. DeltaNp73). It was clearly shown that p73 transactivation from a p53-responsive promoter is inhibited by some tumor-derived p53 mutants in eucaryotic cells. In this study, we adapted a yeast-based p53 functional assay for the analysis of the influences of different p53 mutants on the activity of one of the p73 isoforms, namely p73beta. We determined the ability of a panel of 61 p53 mutants to inhibit p73beta activity following the net transcription of the ADE2 color (red/white) reporter gene driven by a p53-responsive promoter. By analysing a large number of mutants, we could conclude that interference: (a) is a quite frequent phenomenon (more than 70% of p53 mutants analysed are interfering); (b) is not confined to p53 mutations located in particular topological regions of the DNA binding domain; (c) does not appear to be dependent on the kind of side chains introduced at a specific position; (d) appears to significantly correlate with evolutionary conservation of the mutated p53 codon, frequency of occurrence of the mutation in tumors. The influence of a common R/P polymorphism at codon 72 on the ability of p53 mutants to interfere with p73beta was also studied. Two sets of polymorphic variants (R and P) for 14 mutants were constructed and analysed. In all cases, the R/P 72 polymorphism was phenotypically irrelevant. In conclusion, our results suggest that the interpretation of the biological effects of p53 mutants should take into consideration the possibility that p53 mutants show loss or gain of function also through the interference with p53 family members.