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.

Repetitive systemic exposure to high concentrations of curcumin achieved by Theracurmin did not increase the incidence of adverse events in cancer patients receiving gemcitabine-based chemotherapy.

Tumor suppressor p53 forms a homo-tetramer through its COOH-terminal oligomerization domain and acts as a sequence-specific transcription factor. We have analysed the interrelation among the transcriptional activities, the structure and the cancer-related mutations in the oligomerization domain by using a comprehensive missense mutation library. Here, we examined the ability of 184 mutant p53s in the domain to form an oligomer by expressing these mutant p53s in yeast, and compared the data with the previous information. We showed that specific residues in the a-helix and the b-strand of the oligomerization domain were critical for both oligomer formation and sequence-specific transactivation, and the activities were closely related. In particular, the a-helix was more sensitive to amino-acid substitutions than the b-strand. We found identity in the interrelation between the two activities, that is, monomer mutants were transcriptionally inactive whereas dimer and tetramer mutants retained their transcriptional activities. In TP53 mutation databases, a small number of mutations have been reported in this domain. Surprisingly, most do not encode p53s defective in functional properties. These results indicate that, although oligomer formation is essential for p53 transactivation function, the inactivation of oligomer formation and therefore the inactivation of transactivation may not be essential for tumor suppression by p53 because they do not lead to oncogenic proteins.

Tumor suppressor p53-dependent apoptosis is thought to be one of the most important tumor-suppressive functions in human tumorigenesis. However, whether the major mechanism underlying the p53-dependent apoptosis is transactivation dependent or independent remains unclear. Using 179 mutant p53s with diverse transcriptional activities for distinct p53-binding sequences in yeast, we evaluated both their sequence-specific transcriptional activities on six p53 target genes and their ability to induce apoptosis in Saos-2 cells. These mutant p53s also represented diversity in their ability to both transactivate target genes and induce apoptosis. We identified 17 mutant p53s with superior ability to induce apoptosis than wild-type p53 that tend to cluster at residues 121 or 290 to 292. There was no significant correlation between the two functional properties on any single target gene examined. Furthermore, the 17 mutant p53s were not classified in a specific cluster by hierarchical cluster analysis on their diverse transcriptional activities, indicating that these mutant p53s were not similar in the transcriptional activity of downstream genes. These results suggested that transactivation-dependent apoptosis does not always play a major role in p53-dependent apoptosis, indirectly supporting the importance role of the transactivation-independent mechanism. (Cancer Res 2005; 65(6): 2108-14)

Temperature-sensitive (ts) mutations have been used as a genetic and molecular tool to study the functions of many gene products. Each ts mutant protein may contain a temperature-dependent intramolecular mechanism such as ts conformational change. To identify key ts structural elements controlling the protein function, we screened ts p53 mutants from a comprehensive mutation library consisting of 2,314 p53 missense mutations for their sequence-specific transactivity through p53-binding sequences in Saccharomyces cerevisiae. We isolated 142 ts p53 mutants, including 131 unreported ts mutants. were ts hot spots. Among the 142 mutants, 54 were examined further in human osteosarcoma Saos-2 cells, and it was confirmed that 89% of the mutants were also ts in mammalian cells. The ts mutants represented distinct ts transactivities for the p53 binding sequences and a distinct epitope expression pattern for conformation-specific anti-p53 antibodies. These results indicated that the intramolecular ␤-sheet in the core DNA-binding domain of p53 was a key structural element controlling the protein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function.p53 tumor suppressor is a 393-amino acid transcription factor that activates the transcription of a number of downstream genes through p53 binding to two copies of the specific consensus DNA sequence 5Ј-RRRC(A/T)(T/A)GYYY-3Ј (in which R is a purine nucleoside and Y is a pyrimidine nucleoside) in their regulatory regions (1). These molecular switches are activated by post-translational modifications, including phosphorylation, acetylation, and prolyl isomerization (2-5) of p53 in response to genotoxic or non-genotoxic stresses. The resulting biological effects are cell cycle arrest, apoptosis, DNA repair, and angiogenesis (6 -10). A growing number of p53 downstream genes have been isolated, and p53 has been structurally and functionally divided into three portions, namely the NH 2 -terminal portion containing the transactivation domain, the central core portion corresponding to the DNA-binding domain, and the COOH-terminal portion containing the oligomerization domain. The evolution of the DNA-binding domain is highly conserved in p53 orthologues (11) and also in the conserved human homologues p63 and p73 (12, 13).The structure of the DNA-binding domain (residues 94 -312) was resolved by x-ray crystallography (14). The domain consists of two ␣-helixes (H1 and H2) and 11 ␤-strands (S1, S2, S2Ј, and S3-S10) that were interconnected by loops (long L1-L3 loops and other short loops). Two anti-parallel ␤-sheets containing four (S1, S3, S5, and S8) and five (S4, S6, S7, S9, and S10) ␤-strands make up a large ␤-sandwich that serves as a scaffold for a loop-sheet-helix (LSH) motif (L1, S2, S2Ј, S10, and H2) and two large loops (L2 and L3). The loop-sheet-helix consists of two separate regions as follows: (i) the L1 loop (residues 113-123) and the S2-S2Ј ␤-hairpin (residues 124 -135) that correspond to evolutionary conserved regio...