Identification of human p53 mutations with differential effects on the bax and p21 promoters using functional assays in yeast

Abstract

Recent studies have suggested that a rare class of p53 mutants found in tumours has a subtle transcriptional defect aecting bax induction but not p21 induction. We have therefore developed simple functional assays in yeast which can be used to identify these mutants. Analysis of 51 dierent mutations observed in human tumours showed that all mutants tested scored as mutant with the bax reporter strain but nine scored as wild-type with the p21 reporter strain. These results, which can be explained by the lower anity of the p53 protein for the bax site, may suggest that p21 is not the key target of p53 mutations in tumours. Since p21 status has recently been shown to modulate the chemotherapeutic and radiotherapeutic sensitivities of cancerous cells, the functional assays described here may have important clinical implications.

p53 mutants can often transactivate promoters containing a p21 but not Bax or PIG3 responsive elements

“…The likely basis for this di erence is that these mutants have a modestly reduced a nity for DNA, allowing them to bind to promoters with high a nity p53 binding sites, as in the p21 promoter, but not to promoters with low a nity sites, as in the Bax promoter. Using simple yeast functional assays, the same mutants have been shown to be competent for transactivation of p21 but not Bax p53 responsive elements in yeast (Flaman et al, 1998). Thus, the yeast assay can be used to predict the transcriptional behaviour of a mutant in higher eukaryotes.…”

“…Be that as it may, the clear conclusion is that the yeast p53 mutation screening assay will miss a certain number of p53 mutations in familial breast cancer. This, however, is unlikely to be a problem except in rare familial cases, and the consequent loss of sensitivity must be set against the other advantages of the assay for detection of mutations in clinical material (Flaman et al, 1995(Flaman et al, , 1998Waridel et al, 1997;Chappuis et al, 1999;Robert et al, 2000;Duddy et al, 2000). The existence of partially active p53 mutants, the postulated role of transcription-independent p53 functions in promoting tumour growth, and the development of drugs which can stabilize mutant p53 core domains all suggest that there may be situations where knowing the precise mutation in p53 may have clinical value.…”

“…To answer these questions, we examined 77 mutants (Campomenosi et al, 1997;Inga et al, 1997Inga et al, , 1998Inga et al, , 1999Kelly et al, 1999;Monti et al, 2000) that were originally isolated in the yIG397 yeast strain (Flaman et al, 1995), which expresses an ADE2 reporter gene from a p53-responsive promoter containing three copies of the RGC sequence (Kern et al, 1991). The YPH-p21, YPH-bax and YPH-PIG3 reporter strains express ADE2 from promoters containing the following p53 responsive elements: p21, 5'-GAACATGTCCCAACATGTTG-3'; bax, 5'-TCACA-AGTTAGAGACAAGCCTGGGCGTGGGCTATAT-TG-3'; PIG3, 5'-CAGCTTGCCCACCCATGCTC-3' (Flaman et al, 1998;Robert et al, 2000).…”

“…These drugs should be most e ective on tumours with 24¡C 30°C 37¡C Figure 1 Example of the results of the analysis of the mutant temperature sensitivity. In order to analyse whether a mutant was able to transactivate the reporter gene under the control of di erent p53 responsive elements, S.cerevisiae haploid strains [YPH-p21: (p21::pCYC1::ADE2) (Flaman et al, 1998); YPH-bax (bax::pCYC1::ADE2) (Flaman et al, 1998); (YPH-PIG3) (PIG3::pCYC1::ADE2), (Robert et al, 2000)] were transformed by the LiAc method with pLS76 yeast expression vector (CEN6/ ARS4, LEU2) coding for the human wild type (WT) or mutated (170R) p53 cDNA under an ADH1 constitutive promoter. Transformants were selected on synthetic minimal medium (Difco, Milano, Italy) lacking leucine, but containing a limiting amount of adenine (5 mg/l).…”

Characterization of the p53 mutants ability to inhibit p73β transactivation using a yeast-based functional assay

“…Saccharomyces cerevisiae haploid p53-reporter strains, yIG397 (3 Â RGC::pCYC1::ADE2) (Inga et al, 1997b), YPH-p21 and YPH-bax (Flaman et al, 1998), were transformed with pLS76 yeast expression vector (CEN6/ARS4, LEU2) coding for the human wild-type (or mutated) p53 cDNA under an ADH1 promoter. Transformants were selected on synthetic minimal medium (Difco, Milano, Italy) lacking leucine, but containing a limiting amount of adenine (5 mg/l).…”