Kim H, You S, Farris J, Foster L K, Foster D N
Department of Animal Science, University of Minnesota, St. Paul, Minnesota, MN 55108, USA.
Oncogene. 2001 May 31;20(25):3306-10. doi: 10.1038/sj.onc.1204423.
The steady-state levels of p53 mRNA and protein were barely detectable by Northern and Western blot analysis in spontaneously immortalized (10)3 and (10)7 murine embryo fibroblast (MEF) cells. But when cells were treated with cycloheximide (CHX) or emetine, expression levels were restored to those observed in primary and immortal (10)10 MEF cells. However, levels of p53 mRNA were not changed in primary or (10)10 MEF cells by CHX treatment. De novo p53 mRNA synthetic rates were similar in primary, (10)10, (10)3, and (10)7 MEF cells treated with or without CHX. Treatment with actinomycin D (ActD) showed that p53 mRNA in primary and (10)10 MEF cells had a relatively long half-life of 22 h, compared to less than 2 h for (10)3 and (10)7 MEF cells. Pulse-chase analysis of p53 mRNA turnover using CHX and ActD showed that the rapid destabilization of p53 mRNA in (10)3 and (10)7 MEF cells could be regulated at the transcriptional and translational levels. In addition, the destabilization of p53 mRNA appeared to occur in the nucleus for (10)3 and (10)7 cells, but not for primary and (10)10 MEF cells. Taken together, the present study demonstrates that inactivation of the p53 gene occurs at the post-transcriptional level by rapid destabilization of its mRNA in the nucleus of spontaneously immortalized (10)3 and (10)7 MEF cells.
在自发永生化的（10）³和（10）⁷小鼠胚胎成纤维细胞（MEF）中，通过Northern和Western印迹分析几乎检测不到p53 mRNA和蛋白质的稳态水平。但是，当细胞用环己酰亚胺（CHX）或依米丁处理时，表达水平恢复到在原代和永生化的（10）¹⁰ MEF细胞中观察到的水平。然而，CHX处理并未改变原代或（10）¹⁰ MEF细胞中p53 mRNA的水平。在用或不用CHX处理的原代、（10）¹⁰、（10）³和（10）⁷ MEF细胞中，p53 mRNA的从头合成速率相似。用放线菌素D（ActD）处理表明，原代和（10）¹⁰ MEF细胞中的p53 mRNA半衰期相对较长，为22小时，而（10）³和（10）⁷ MEF细胞的半衰期不到2小时。使用CHX和ActD对p53 mRNA周转进行脉冲追踪分析表明，（10）³和（10）⁷ MEF细胞中p53 mRNA的快速去稳定化可在转录和翻译水平上受到调控。此外，p53 mRNA的去稳定化似乎发生在（10）³和（10）⁷细胞的细胞核中，但在原代和（10）¹⁰ MEF细胞中则不然。综上所述，本研究表明，在自发永生化的（10）³和（10）⁷ MEF细胞的细胞核中，p53基因通过其mRNA的快速去稳定化在转录后水平发生失活。
