Reich N C, Oren M, Levine A J
Mol Cell Biol. 1983 Dec;3(12):2143-50. doi: 10.1128/mcb.3.12.2143-2150.1983.
The steady-state levels of p53 protein and p53 mRNA in transformed and nontransformed cells were examined to elucidate the mechanisms controlling expression of p53. mRNA levels were determined by Northern blot hybridization analysis, employing a p53-specific cDNA clone (M. Oren and A.J. Levine, Proc. Natl. Acad. Sci. U.S.A. 80:56-59, 1983), and protein levels were determined by the Western blotting technique. Analysis of p53 mRNA revealed a single polyadenylated mRNA species migrating at ca. 18S. Levels of p53 mRNA in simian virus 40-transformed cell line (SVT2) and in an homologous nontransformed cell line (3T3) were equivalent, although the steady-state levels of p53 protein were 25- to 100-fold higher in the SVT2 cells than in the 3T3 cells. A study with a non-virus-transformed cell system revealed a different result. Embryonal carcinoma cells (F9) were found to have nearly 20-fold higher levels of p53 mRNA in comparison with differentiated benign progeny cells. In this system the difference in p53 mRNA levels corresponded to the difference in p53 protein levels. Pulse-chase experiments were performed to study the half-life of p53 protein in these four types of cells. The turnover of p53 protein occurred with biphasic kinetics. In addition, it was found that protein synthesis inhibitors placed in the medium during the chase period prevented the turnover of p53 protein in transformed cells, but not in nontransformed (3T3) cells. These results provide evidence that the regulation of p53 expression in cells can occur at the level of p53 mRNA abundancy or p53 protein stability depending upon the experimental system under study, and that a regulated degradation process controls the turnover of p53 protein.
检测了转化细胞和未转化细胞中p53蛋白和p53 mRNA的稳态水平，以阐明控制p53表达的机制。采用p53特异性cDNA克隆（M. 奥伦和A.J. 莱文，《美国国家科学院院刊》80:56 - 59, 1983），通过Northern印迹杂交分析确定mRNA水平，并用蛋白质印迹技术确定蛋白质水平。对p53 mRNA的分析显示，有一个单一的多聚腺苷酸化mRNA条带，迁移率约为18S。猿猴病毒40转化细胞系（SVT2）和同源未转化细胞系（3T3）中p53 mRNA的水平相当，尽管SVT2细胞中p53蛋白的稳态水平比3T3细胞高25至100倍。一项对非病毒转化细胞系统的研究得出了不同的结果。发现胚胎癌细胞（F9）的p53 mRNA水平比分化的良性后代细胞高近20倍。在这个系统中，p53 mRNA水平的差异与p�3蛋白水平的差异相对应。进行脉冲追踪实验以研究这四种类型细胞中p53蛋白的半衰期。p53蛋白的周转呈现双相动力学。此外，还发现，在追踪期将蛋白质合成抑制剂加入培养基中，可阻止转化细胞中p53蛋白的周转，但不能阻止未转化（3T3）细胞中p53蛋白的周转。这些结果证明，细胞中p53表达的调控可根据所研究的实验系统，在p53 mRNA丰度水平或p53蛋白稳定性水平上发生，并且一个受调控的降解过程控制着p53蛋白的周转。
