| Summary: | 碩士 === 高雄醫學大學 === 醫學研究所-基礎醫學組 === 102 === TP53 mutations are common in various tumors. Many of these mutations affect p53-mediated transcriptional regulation. Previous studies show that p53 lysine (K) mutations alter expression of downstream genes including apoptosis- and cell cycle-related genes. However, whether p53 K mutations affect expression of DNA repair genes DDB2 and XPC is unknown. This study focuses on the effects of various p53 K mutations on control of downstream gene expression, especially on the DDB2 and XPC. We used site-direct mutagenesis to construct p53 K mutant plasmids: K120R, K120M, K120Q, K120A, 6KR (K370R, K372R, K373R, K381R, K382R, K386R), K120R/6KR, K120M/6KR, K164/6KR, 8KR (K120R/K164R/6KR), all were verified by DNA sequencing and checked by Western blot for protein expression and post-translational modifications (acetylation, methylation, phosphorylation) in the p53-null H1299 cells. The effects of these K mutants on transactivation of downstream genes were examined and compared with that of wild type (WT) p53 by using reporter assay and quantitative RT-PCR (qRT-PCR). The results showed that all of these p53 mutants could be expressed, as well as the WT p53, in H1299. The C-terminal K mutant 6KR preserved transactivation capacity as that of WT p53 on the examined downstream genes. Mutation at K120 resulted in increases of acetylation at K305, K373, and K382. However, K120 mutants exhibited a decreased transactivation activity toward to the CDKN1A promoter and led to a less induction in the CDKN1A RNA expression when compared with WT p53. The effects of K120 mutantions on DDB2 promoter were less than those on CDKN1A in reporter assays. Based on the results of qRT-PCR, K120Q, likes as WT, could activate DDB2, XPC, and GADD45 RNA expression. But K120R and K120M showed a reduced abitlity in the activation of DDB2, XPC, BAX, GADD45, and GLS2 RNA expression. The effects of arecoline (the major alkaline of betel nut) and adriamycin (an anticancer drug) on the transactivation activities of these p53 mutants were also examined. Together, this study shows that mutations of p53 K120 cause changes of acetylation at other residues and alterations of several downstream gene expressions.
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