| Co-Investigator(Kenkyū-buntansha) |
SAKAYORI Masato Tohoku University, Hospital, Research Associate, 病院・助手 (20344666)
KATO Shunsuke Tohoku University, Hospital, Research Associate, 病院・助手 (60333887)
SHIBATA Hiroyuki Tohoku University, Hospital, Lecturer, 病院・講師 (50260071)
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| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2003: ¥5,400,000 (Direct Cost: ¥5,400,000)
Fiscal Year 2002: ¥8,600,000 (Direct Cost: ¥8,600,000)
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| Research Abstract |
The objectives of this study were (i) development of p53-associated molecular devices for diagnosis of gastrointestinal cancer, including novel method of detecting p53 gene mutations and mutant p53 proteins, and (ii) development of p53-associated molecular devices for treatment of gastrointestinal cancer, including improved p53 cDNA for p53-mediated gene therapy and small molecule compounds for restoration of inactivated mutant p53 function. We developed several important p53-related molecular devices. First, by using a comprehensive site-directed mutagenesis technique and a yeast-based functional assay to construct, express, and evalute 2,314 p53 mutants representing all possible amino acid substitutions caused by a point mutation throughout the protein (5.9 substitutions per residue), and cerrelated p53 function with structure-and tumor-derived mutations. Second, to identify key temperature-sensitive (ts) structural elements controlling the protein function, we screened ts p53 mutant
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s 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 and showed that the intramolecular beta-sheet in the core DNA-binding domain of p53 was a key structural element controlling the rotein function and provided a clue for finding a molecular mechanism that enables the rescue of the mutant p53 function. Third, by using the p53 mutation library, we identified second-site suppressor (sss) mutations for common p53 mutations. The identified sss mutations restored two of the common mutations by both intramolecular and intermolecular manner. The results suggested that inactivated function of specific p53 mutants might be recovered by extramolecules. Fourth, as a result of intensive screening of the p53 mutation library, we identified at least several mutant p53 that had the stronger activity to induce apoptosis than wild-type p53. These mutant p53 might be useful cDNA resources for cancer gene therapy. Finally, we are screening in silico of small molecular compounds that might restore the inactivated function of mutant p53 through a molecular docking simulation method, and found several candidate molecules. Less
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