Characterization of the p53 mutants ability to inhibit p73β 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 transcriptionincompetent forms (i.e. DNp73). 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 p73b. We determined the ability of a panel of 61 p53 mutants to inhibit p73b 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 p73b 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.

The prevalence of germ-line TP53 mutations in women diagnosed with breast cancer before age 30

“…This mutation has been previously reported as a somatic mutation in breast, ovary, kidney, and stomach cancers [12][13][14]. The functional effects of missense mutations for codon 283 have been confirmed [15][16][17][18][19][20][21][22][23]. The proband, of Italian ethnic origin, was diagnosed with breast cancer at age 29 and with ovarian cancer at age 56 (Fig.…”

Structure of p73 DNA-binding domain tetramer modulates p73 transactivation

“…In the assay, p73 REs are used as upstream enhancers of the expression of a firefly luciferase gene controlled by a minimal promoter and cloned into a constant chromosomal location in isogenic yeast strains that naturally do not contain a p73 homolog (13). In experiments with yeast cells, we previously demonstrated that human p73β protein can act as a transcription factor using constitutive and inducible promoters (29). Hence, we tested the ability of human p73β to induce the expression of the luciferase gene under the enhancer control of 10 REs with spacers from 0 to 4 bp that are variations of the three half-site sequences used for crystallization (Fig.…”

Analysis of germline and tumor mutations of p53 gene in familial occurrence of soft tissue sarcomas

“…It is one of hotspots in p53 mutations and has a dominant-negative activity. On the contrary, the mutation in codon 270 (exon 8) in Case 2 is extremely rare and there have been very few reports on it [17,18]. This mutation has not dominant-negative activity [17] and inhibits recessively transactivation of p73 by binding to p73 [18].…”

“…On the contrary, the mutation in codon 270 (exon 8) in Case 2 is extremely rare and there have been very few reports on it [17,18]. This mutation has not dominant-negative activity [17] and inhibits recessively transactivation of p73 by binding to p73 [18]. Narita et al described germline mutations of p53 at exon 4-9 were not detected in a female patient with malignant fibrous histiocytoma, whose son and daughter developed liposarcomas [8].…”