Center of Molecular Biology and Gene Therapy, Department of Internal Medicine - Hematooncology, University Hospital Brno and Faculty of Medicine, Masaryk University, Cernopolni 9, CZ-625 00 Brno, Czech Republic.
Loschmidt Laboratories, Institute of Experimental Biology and National Center for Biomolecular Research, Faculty of Science, Masaryk University, Kamenice 5/A4, CZ-625 00 Brno, Czech Republic.
Biol Chem. 2010 Feb-Mar;391(2-3):197-205. doi: 10.1515/bc.2010.027.
Sequence-specific DNA binding is the key function through which tumor suppressor p53 exerts transactivation of the downstream target genes, often being impaired in cancer cells by mutations in the TP53 gene. Functional protein microarray technology enables a high-throughput parallel analysis of protein properties within one experiment under the same conditions. Using an array approach, we analyzed the DNA binding activity of wild type p53 protein and of 49 variants. Our results show significant differences in the binding properties between the p53 mutants. The C-terminal mutant R337C displayed the highest DNA binding activity on the array. However, the same mutant showed only a partial activation in the reporter gene assay and almost no activation of downstream target genes after transfection of expression vector into cells lacking endogenous p53. These observations demonstrate that DNA binding itself is not sufficient for activating the p53 target genes in at least some of the p53 mutants and, therefore, in vitro studies might not always reflect in vivo conditions.
序列特异性 DNA 结合是肿瘤抑制因子 p53 发挥下游靶基因转录激活的关键功能，TP53 基因突变常导致癌细胞中这一功能受损。功能性蛋白质微阵列技术可在相同条件下，在一次实验中同时高通量平行分析蛋白质的特性。我们使用阵列方法分析了野生型 p53 蛋白和 49 种变体的 DNA 结合活性。结果表明，p53 突变体之间的结合特性存在显著差异。C 端突变体 R337C 在阵列上表现出最高的 DNA 结合活性。然而，在报告基因检测中，相同的突变体只显示部分激活，并且在缺乏内源性 p53 的细胞中转染表达载体后，下游靶基因几乎没有激活。这些观察结果表明，DNA 结合本身不足以激活至少部分 p53 突变体中的 p53 靶基因，因此体外研究可能并不总是反映体内条件。
