Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function
Targeted point mutations of p53 lead to dominant-negative inhibition of wild-type p53 function
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DOI：
10.1073/pnas.052713099
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发表时间：
2002-03-05
影响因子：
11.1
通讯作者：
Jacks, T
中科院分区：
文献类型：
--
作者：
de Vries, A;Flores, ER;Jacks, T
p53肿瘤抑制基因是人类癌症中最常见的突变基因，并且生殖系p53突变导致癌症的家族易感性。生殖系或散发性p53突变通常是错义的，并且通常影响蛋白质的中心DNA结合结构域。由于p53作为四聚体转录因子发挥作用，突变型p53被认为抑制野生型p53蛋白的功能。在这里，我们研究了可能的显性负抑制野生型p53蛋白的两个不同的，经常发生的点突变。将R270 H和P275 S突变靶向小鼠胚胎干细胞的基因组中，以允许分析在单拷贝水平下在正常细胞中表达的突变蛋白的影响。在胚胎干细胞中，一个杂合子点突变等位基因的存在导致延迟转录激活的几个p53下游靶基因暴露于γ射线。与野生型细胞相比，突变胚胎干细胞中阿霉素诱导的凋亡受到严重影响。杂合子突变胸腺细胞有一个严重的缺陷，在p53依赖的凋亡途径后，与γ射线或阿霉素治疗，而p53独立的凋亡途径是完整的。总之，这些数据表明，点突变的p53的生理表达可以强烈限制整体细胞p53功能，支持这种突变体的显性负作用。此外，对于此类突变杂合的细胞可能在肿瘤抑制和对化疗剂的反应方面受损。
The p53 tumor suppressor gene is the most frequently mutated gene in human cancers, and germ-line p53 mutations cause a familial predisposition for cancer. Germ-line or sporadic p53 mutations are usually missense and typically affect the central DNA-binding domain of the protein. Because p53 functions as a tetrameric transcription factor, mutant p53 is thought to inhibit the function of wild-type p53 protein. Here, we studied the possible dominant-negative inhibition of wild-type p53 protein by two different, frequently occurring point mutations. The R270H and P275S mutations were targeted into the genome of mouse embryonic stem cells to allow the analysis of the effects of the mutant proteins expressed in normal cells at single-copy levels. In embryonic stem cells, the presence of a heterozygous point-mutated allele resulted in delayed transcriptional activation of several p53 downstream target genes on exposure to gamma irradiation. Doxorubicin-induced apoptosis was severely affected in the mutant embryonic stem cells compared with wild-type cells. Heterozygous mutant thymocytes had a severe defect in p53-dependent apoptotic pathways after treatment with gamma irradiation or doxorubicin, whereas p53-independent apoptotic pathways were intact. Together these data demonstrate that physiological expression of point-mutated p53 can strongly limit overall cellular p53 function, supporting the dominant-negative action of such mutants. Also, cells heterozygous for such mutations may be compromised in terms of tumor suppression and response to chemotherapeutic agents.