Grand R J, Lecane P S, Owen D, Grant M L, Roberts S, Levine A J, Gallimore P H
CRC Institute for Cancer Studies, Medical School, University of Birmingham, Edgbaston, United Kingdom.
Virology. 1995 Jul 10;210(2):323-34. doi: 10.1006/viro.1995.1349.
The cellular protein MDM2 can bind to the tumor suppressor gene product p53 and abrogate its transcriptional activity. In addition, p53 can regulate expression of the mdm2 gene. We and others have previously shown that p53 is present at high levels in adenovirus-transformed cells which express the larger E1B protein. In view of these observations the expression of MDM2 in a panel of adenovirus transformed human cell lines has been examined. Two major species (98K and 80K) were detected, together with a number of minor species of higher and lower molecular weight. While there was little variation in levels of 98K protein between cell lines, appreciable differences in the expression of the 80K component were apparent. There was no correlation between MDM2 and p53 expression in any of the adenovirus transformants, nor with the viral proteins expressed. The pattern and level of MDM2 detected was similar to that seen in human tumor cell lines and in human fetal tissue. Northern blot analysis suggested that MDM2 expression was regulated at the transcriptional level. Stable interactions were observed between p53 and MDM2 in the adenovirus-transformed cell lines and in Ad5 E1 HEK 293 cells a ternary complex of p53, MDM2, and the Ad5 E1B 58K protein was demonstrated. In view of the lack of correlation between the level of p53 and MDM2 in adenovirus E1-transformed cells, the capacity of p53 to cause transcriptional activation was assessed using transfected CAT constructs linked to p53 responsive elements. p53 transcriptional activity was similar in all of the cell lines examined and did not correlate with protein expression. It is concluded, on the basis of all of these data, that the high concentrations of p53 found in adenovirus transformants are not transcriptionally active and have no influence on MDM2 expression. However, when expression of p53 was increased following infection with mutant adenoviruses, which do not express the larger E1B proteins, there was an appreciable increase in p53 transcriptional activity and in the levels of all of the MDM2 components.
细胞蛋白MDM2可与肿瘤抑制基因产物p53结合并消除其转录活性。此外，p53可调节mdm2基因的表达。我们和其他人之前已经表明，p53在表达较大E1B蛋白的腺病毒转化细胞中含量很高。鉴于这些观察结果，我们检测了一组腺病毒转化的人类细胞系中MDM2的表达。检测到两个主要条带（98K和80K），以及一些分子量更高和更低的次要条带。虽然细胞系之间98K蛋白的水平变化不大，但80K组分的表达存在明显差异。在任何腺病毒转化体中，MDM2和p53的表达之间均无相关性，与所表达的病毒蛋白也无相关性。检测到的MDM2的模式和水平与在人类肿瘤细胞系和人类胎儿组织中观察到的相似。Northern印迹分析表明MDM2的表达在转录水平受到调节。在腺病毒转化的细胞系中观察到p53和MDM2之间存在稳定的相互作用，并且在Ad5 E1 HEK 293细胞中证实了p53、MDM2和Ad5 E1B 58K蛋白的三元复合物。鉴于腺病毒E1转化细胞中p53和MDM2水平之间缺乏相关性，使用与p53反应元件相连的转染CAT构建体评估了p53引起转录激活的能力。在所检测的所有细胞系中，p53的转录活性相似，并且与蛋白表达无关。基于所有这些数据得出的结论是，在腺病毒转化体中发现的高浓度p53没有转录活性，并且对MDM2的表达没有影响。然而，当用不表达较大E1B蛋白的突变腺病毒感染后p53的表达增加时，p53的转录活性以及所有MDM2组分的水平都有明显增加。
