The transcription factor and tumor suppressor protein p53 is frequently inactivated in human cancers. In many cases, p53 gene mutations result in high levels of inactive, full-length p53 protein with one amino acid change in the core domain that recognizes p53 DNA-binding sites. The ability to endow function to mutated p53 proteins would dramatically improve cancer therapy, because it would reactivate a central apoptotic pathway. By using genetic strategies and p53 assays in yeast and mammalian cells, we identified a global suppressor motif involving codons 235, 239, and 240. These intragenic suppressor mutations, either alone or in combination, restored function to 16 of 30 of the most common p53 cancer mutants tested. The 235-239 -240 suppressor motif establishes that manipulation of a small region of the core domain is sufficient to activate a large number of p53 cancer mutants. Understanding the structural basis of the rescue mechanism will allow the pursuit of small compounds able to achieve a similar stabilization of p53 cancer mutants.

Over 1000 different mutants of the tumor suppressor protein p53 with one amino acid change in the core domain have been reported in human cancers. In mouse knock-in models, two frequent mutants displayed loss of wild-type (wt) p53 function, inhibition of wt p53 and wt p53-independent gain of function. The remaining mutants have been systematically characterized for loss of wt p53 function, but not other phenotypes. We report the concomitant assessment of loss of function and interference with wt p53 using URA3-based p53 yeast and confirmatory mammalian assays. We studied 76 mutants representing 54% of over 15 000 reported missense core domain mutations. The majority showed the expected complete loss of wt p53 function and dominant p53 inhibition. A few infrequent p53 mutants had wt p53-like activity. Remarkably, one-third showed no interference with wt p53 despite loss of wt p53 function at 37 degrees C. Half of this group consisted of temperature-sensitive p53 mutants, but the other half was surprisingly made up of mutants with complete loss of wt p53 function. Our findings illustrate the diverse behavior of p53 mutants and mechanisms of malignant transformation by p53 mutants. The identification of full-length p53 mutants without dominant inhibition of wt p53 highlights the importance of determining the status of the wt p53 allele in human cancers, in particular in the context of clinical studies. In the case of p53 mutants with no or weak dominant p53 inhibition, presence of the wt allele may indicate a good prognosis cancer, whereas loss of heterozygosity may spell an aggressive, therapy-resistant cancer.

Many biomedical problems relate to mutant functional properties across a sequence space of interest, e.g., flu, cancer, and HIV. Detailed knowledge of mutant properties and function improves medical treatment and prevention. A functional census of p53 cancer rescue mutants would aid the search for cancer treatments from p53 rescue. We devised a general methodology for conducting a functional census of a mutation sequence space, and conducted a double-blind predictive test on the functional rescue property of 71 novel putative p53 cancer rescue mutants iteratively predicted in sets of 3. Double-blind predictive accuracy (15-point moving window) rose from 47% to 86% over the trial (r = 0.74). Code and data are available upon request1.

Previous work from our laboratory demonstrated that CCAAT/enhancer-binding protein ␦ (C/EBP␦) functions in the initiation and maintenance of G 0 growth arrest in mouse mammary epithelial cells (MECs). In this report, we investigated the posttranscriptional and posttranslational regulation of C/EBP␦ in G 0 growth-arrested mouse MECs. The results of transcriptional inhibitor studies demonstrated that the C/EBP␦ mRNA exhibits a relatively short half-life in G 0 growth-arrested mouse MECs (t1 ⁄2 ϳ ϳ35 min). In contrast, C/EBP␦ mRNA has a longer half-life in G 0 growth-arrested mouse fibroblast cells (t1 ⁄2 >100 min). Oligo/RNase H cleavage analysis and rapid amplification of cDNA ends-poly(A) test both confirmed the short C/EBP␦ mRNA half-life observed in MECs and demonstrated that the C/EBP␦ mRNA poly(A) tail is relatively short (ϳ ϳ100 nucleotides). In addition, the poly(A) tail length was not shortened during C/EBP␦ mRNA degradation, which suggested a deadenylationindependent pathway. The C/EBP␦ protein also exhibited a relatively short half-life in G 0 growth-arrested mouse MECs (t1 ⁄2 ϳ ϳ120 min). The C/EBP␦ protein was degraded in a ubiquitin-dependent manner, primarily in the nucleus, during G 0 growth arrest. In conclusion, these studies indicated that the C/EBP␦ mRNA and protein content are under tight regulation in G 0 growtharrested mouse MECs, despite the general concept that G 0 growth arrest is associated with a decrease in cellular activity.