Communicated by David GoldgarThe tumor suppressor gene TP53 is frequently mutated in human cancers. More than 75% of all mutations are missense substitutions that have been extensively analyzed in various yeast and human cell assays. The International Agency for Research on Cancer (IARC) TP53 database (www-p53.iarc.fr) compiles all genetic variations that have been reported in TP53. Here, we present recent database developments that include new annotations on the functional properties of mutant proteins, and we perform a systematic analysis of the database to determine the functional properties that contribute to the occurrence of mutational ''hotspots'' in different cancer types and to the phenotype of tumors. This analysis showed that loss of transactivation capacity is a key factor for the selection of missense mutations, and that difference in mutation frequencies is closely related to nucleotide substitution rates along TP53 coding sequence. An interesting new finding is that in patients with an inherited missense mutation, the age at onset of tumors was related to the functional severity of the mutation, mutations with total loss of transactivation activity being associated with earlier cancer onset compared to mutations that retain partial transactivation capacity. Furthermore, 80% of the most common mutants show a capacity to exert dominant-negative effect (DNE) over wild-type p53, compared to only 45% of the less frequent mutants studied, suggesting that DNE may play a role in shaping mutation patterns. These results provide new insights into the factors that shape mutation patterns and influence mutation phenotype, which may have clinical interest. Hum Mutat 28(6), 622-629, 2007. Published 2007

Inactivation of the tumor suppressor p53 by missense mutations is the most frequent genetic alteration in human cancers. The common missense mutations in the TP53 gene disrupt the ability of p53 to bind to DNA and consequently to transactivate downstream genes. However, it is still not fully understood how a large number of the remaining mutations affect p53 structure and function. Here, we used a comprehensive site-directed mutagenesis technique and a yeastbased functional assay to construct, express, and evaluate 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and correlated p53 function with structure-and tumor-derived mutations. This high-resolution mutation analysis allows evaluation of previous predictions and hypotheses through interrelation of function, structure and mutation.

The p53 tumor suppressor gene, which is induced by DNA damage and/or stress stimuli, causes cells to undergo G1-arrest or apoptotic death; thus it plays an essential role in human carcinogenesis. We have searched for p53-related genes by using degenerate PCR, and have identified two cDNA fragments similar to but distinct from p53: one previously reported, p73, and the other new. We cloned two major splicing variants of the latter gene and named these p51A and p51B (a human homologue of rat Ket). The p51A gene encodes a 448-amino-acid protein with a molecular weight of 50.9 kDa; and p51B, a 641-amino-acid protein with a molecular weight of 71.9 kDa. In contrast with the ubiquitous expression of p53, expression of p51 mRNA was found in a limited number of tissues, including skeletal muscle, placenta, mammary gland, prostate, trachea, thymus, salivary gland, uterus, heart and lung. In p53-deficient cells, p51A induced growth-suppression and apoptosis, and upregulated p21waf-1 through p53 regulatory elements. Mutations in p51 were found in some human epidermal tumors.

To investigate the clinical value of somatic TP53 mutations in breast cancer, we assembled clinical and molecular data on 1,794 women with primary breast cancer with long-term follow-up and whose tumor has been screened for mutation in exons 5 to 8 of TP53 by gene sequencing. TP53 mutations were more frequent in tumors of ductal and medullar types, aggressive phenotype (high grade, large size, node positive cases, and low hormone receptor content) and in women <60 years old. TP53 mutations within exons 5 to 8 conferred an elevated risk of breast cancer^specific death of 2.27 (relative risk >10 years; P < 0.0001) compared with patients with no such mutation. The prognostic value of TP53 mutation was independent of tumor size, node status, and hormone receptor content, confirming and reconciling previous findings in smaller series. Moreover, an interaction between TP53 mutation and progesterone receptor (PR) status was revealed, TP53 mutation combined with the absence of progesterone receptor being associated with the worst prognosis. Whereas previous studies have emphasized the fact that missense mutations in the DNA-binding motifs have a worse prognosis than missense mutations outside these motifs, we show that non^missense mutations have prognostic value similar to missense mutations in DNA-binding motifs. Nonetheless, specific missense mutants (codon 179 and R248W) seem to be associated with an even worse prognosis. These results, obtained on the largest series analyzed thus far, show that TP53 mutations identified by gene sequencing have an independent prognostic value in breast cancer and could have potential uses in clinical practice.The tumor suppressor gene TP53 plays a key role in many cellular pathways controlling cell proliferation, cell survival, and genomic integrity. It acts as a proliferation brake when cells experience stress conditions, such as DNA-damage, hypoxia, or oncogene activation. Disrupting TP53 function promotes checkpoint defects, genomic instability, and inappropriate survival, leading to the uncontrolled proliferation of damaged cells. The proliferative advantage given by its inactivation, and the fact that it is ubiquitously expressed, explains why it is frequently found to be mutated in almost every type of cancer (1). It has been shown in various experimental in vitro systems, as well as in mouse models, that cell cycle arrest or apoptosis induced by radiotherapy and various chemotherapeutic drugs depends on an intact TP53 pathway (2, 3). These results have raised the hypothesis that TP53 could be a key player in defining tumor sensitivity to a broad range of anticancer treatments in patients with cancer. Moreover, the presence of a TP53 mutation could be one of the underlying causes of drug resistance, the major cause of treatment failure and cancer death.Several studies have investigated the predictive value of TP53 mutation status for tumor response to treatment and patient outcome in various cancers. Different clinical and methodologic

Prediction of the biological effect of missense substitutions has become important because they are often observed in known or candidate disease susceptibility genes. In this paper, we carried out a 3-step analysis of 1514 missense substitutions in the DNA-binding domain (DBD) of TP53, the most frequently mutated gene in human cancers. First, we calculated two types of conservation scores based on a TP53 multiple sequence alignment (MSA) for each substitution: (i) Grantham Variation (GV), which measures the degree of biochemical variation among amino acids found at a given position in the MSA; (ii) Grantham Deviation (GD), which reflects the ‘biochemical distance’ of the mutant amino acid from the observed amino acid at a particular position (given by GV). Second, we used a method that combines GV and GD scores, Align-GVGD, to predict the transactivation activity of each missense substitution. We compared our predictions against experimentally measured transactivation activity (yeast assays) to evaluate their accuracy. Finally, the prediction results were compared with those obtained by the program Sorting Intolerant from Tolerant (SIFT) and Dayhoff's classification. Our predictions yielded high prediction accuracy for mutants showing a loss of transactivation (∼88% specificity) with lower prediction accuracy for mutants with transactivation similar to that of the wild-type (67.9 to 71.2% sensitivity). Align-GVGD results were comparable to SIFT (88.3 to 90.6% and 67.4 to 70.3% specificity and sensitivity, respectively) and outperformed Dayhoff's classification (80 and 40.9% specificity and sensitivity, respectively). These results further demonstrate the utility of the Align-GVGD method, which was previously applied to BRCA1. Align-GVGD is available online at .

Effects of breast cancer-associated gene 1 (BRCA1) missense mutations on the function of BRCA1 protein in DNA recombination have been little studied. In this report, we adapted a homology-directed recombination (HDR) assay to analyze the effects of BRCA1 mutations on this function. Using a HeLa-derived cell line with a genomically integrated recombination substrate, we expressed an endonuclease creating a double-stranded break in the substrate that the HDR assay scores by generation of green fluorescent protein-positive cells. By combining RNA interference (RNAi) that targets cellular BRCA1 mRNA with expression of RNAi-resistant BRCA1 mutants, we could effectively substitute selected point mutants to test these in the cellular recombination assay. We found that ∼300 residues at both termini of the BRCA1 protein were essential for HDR. Whereas some mutations analyzed were neutral, mutations that altered any zinc-coordinating residue or generated M18T and T37R alterations were defective for recombination. This study established a robust assay system to analyze the function of BRCA1 in regulating homologous recombination, which is critical for its tumor suppressor function. Cancer Res; 70(3); 988-95. ©2010 AACR.

Inherited mutations of the p53 gene significantly increase the risk of developing diverse malignancies, and germline p53 mutations can be detected by assaying the transcriptional activity of the p53 protein in mammalian cells. Here we describe a method starting with lymphocytes that allows detection of germline p53 mutations by 'functional' analysis of p53 protein expressed in Saccharomyces cerevisiae. The p53 PCR products are directly cloned into yeast expression vectors in vivo and subsequently tested for transcriptional activity in a simple growth assay. This technique, functional analysis of separated alleles in yeast (FASAY), requires only a few steps, can be automated readily and should permit screening for germline or somatic heterozygous mutations in any gene whose function can be monitored in yeast.

Curcumin (diferuloylmethane) is a dietary phytochemical with low toxicity that exhibits growth-suppressive activity against a variety of cancer cells and possesses certain chemopreventive properties. Curcumin has already been the subject of several clinical trials for use as a treatment in human cancers. Synthetic chemical modifications of curcumin have been studied intensively in an attempt to find a molecule with similar but enhanced properties of curcumin. In this study, a series of novel curcumin analogues were synthesized and screened for anticancer activity. New analogues that exhibit growth-suppressive activity 30 times that of curcumin and other commonly used anticancer drugs were identified. Structurally, the new analogues are symmetrical 1,5-diarylpentadienone whose aromatic rings possess an alkoxy substitution at each of the positions 3 and 5. Analysis of the effects of the analogues on the expression of cancer-related genes usually affected by curcumin indicated that some induced the down-regulation of B-catenin, Ki-ras, cyclin D1, c-Myc, and ErbB-2 at as low as one eighth the concentration at which curcumin normally has an effect. The analogues,