Novel human p53 mutations that are toxic to yeast can enhance transactivation of specific promoters and reactivate tumor p53 mutants

Since highly expressed human p53 can inhibit human and yeast cell growth, w e predicted that p53 mutants could be generated with increased growth inhib ition of the yeast Saccharomyces cerevisiae and that these would be useful for characterizing p53 functions and tumor p53 mutants. A random mutagenesi s screen led to the isolation of mutations in the DIVA binding domain that result in p53 being lethal even at moderate expression levels in yeast. Thr ee independent mutants had an alanine change at the evolutionary invariant V122 in the L1 loop. The other toxic mutations affected codons 277 (C277R, C277W) and 279 (G279R). This latter amino acid change was also reported in tumors, while all the other mutations are novel. A recently developed rheos tatable GALI promoter system that provides graded increases in expression o f p53 was used to examine the transactivation function of the toxic mutatio ns when expression was greatly reduced and cells were viable. At low expres sion levels the toxic mutants lacked transactivation from a 3xRGC responsiv e element (RE). Surprisingly some exhibited enhanced transactivation with p 21 and bax REs. The V122A mutant was able to re-activate transactivation of various p53 tumor mutants and retained growth inhibition when co-expressed with dominant-negative tumor mutations. Upon expression in human Saos-2 ce lls the V122A p53 mutant caused growth suppression, was capable of transact ivation and exhibited higher than wild type activity with the bax promoter in luciferase assays. A non-functional p53 tumor mutant was partially react ivated by V122A for both transactivation and growth suppression. Thus, the screen for toxic p53 mutants in yeast can identify novel p53 variants that may be useful in dissecting p53 regulated cellular responses and in develop ing p53-based cancer therapies.