Selection of cell death-deficient p53 mutants in Saccharomyces cerevisiae.

The budding yeast Saccharomyces cerevisiae is a useful system for the detection and transcriptional evaluation of mutant p53 in cancer. In previous work we showed that the overexpression of wild-type p53 induces yeast cell death on minimal medium; however, the R248W p53 mutant was completely inactive, and we suggested that ROS production is a key event in p53-induced yeast cell death. In this study we explored the effect of other p53 mutants, such as the hot-spot mutant R282W and the double mutant N268S::I332V. Unexpectedly, both mutants behaved inversely to R248W, as they completely inhibited yeast growth on minimal medium and induced ROS production. This phenotype 'yeast cell death on minimal medium' allowed for the subsequent screening of intragenic p53-inactivating mutations. In all cases, the 'revertant yeast clones' display a complete p53 inactivation through either gross deletion or nonsense mutations. More interestingly, missense mutations were also found: the deletion of I255 or substitution of R337G completely inactivated the p53 mutant R282W in the yeast context. Taken together, these results suggest that p53 tumour-derived mutants could be classified according to their ability to induce yeast cell death and not uniquely by their transcriptional activity on a selected target reporter gene.

Human p53 induces cell death and downregulates thioredoxin expression in Saccharomyces cerevisiae.

The p53 tumour suppressor protein has a crucial role in controlling cell cycle and apoptosis in human cells and its inactivation by selective point mutations is associated with human cancers. Here we show that overexpression of the human wild-type (wt) p53 in Saccharomyces cerevisiae completely inhibits yeast growth under minimal media conditions. In contrast, the R248W 'hot spot' p53 mutant (one of the most frequent p53 mutations encountered in human cancers) does not impair yeast growth. Moreover, we report, for the first time, that the human wt p53 induces yeast cell death with characteristic markers of apoptosis: exposure of phosphatidylserine and DNA strand cleavage as shown by Annexin V staining and terminal deoxynucleotidyl transferase-mediated dUTP nick end labelling assay, respectively. In addition, p53 also has an impact on the expression of yeast genes. Using differential display and Northern blot analysis, we demonstrated that human wt p53 expression in yeast leads to gene repression of thioredoxin (TRX1/2), a highly conserved multifunctional antioxidative and antiapoptotic protein family. Accordingly, we demonstrated that reactive oxygen species (ROS) are highly produced in p53 yeast induced cell death as shown by dihydrorhodamine 123 staining. These results suggest that the generation of ROS is a key event in p53 yeast induced cell death.