Gudas J M, Oka M, Diella F, Trepel J, Cowan K H
Medical Breast Cancer Section, National Cancer Institute, Bethesda, Maryland 20892.
Cell Growth Differ. 1994 Mar;5(3):295-304.
In this study, we compare the expression patterns of p53 mRNA and protein in normal human mammary epithelial cells following synchronization to different points in the cell cycle using two independent methods. When treated with lovastatin, the cells were blocked in G1 and appeared to express increased levels of wild-type p53 when examined by immunostaining. Upon reversal of the metabolic block, the number of nuclei that stained positively for p53 declined dramatically during mid-G1 and increased again concomitant with the entry of cells into S phase. In contrast to the immunostaining results, Northern and Western blot analyses revealed little change in p53 mRNA and protein levels in the lovastatin-synchronized cells. When normal human mammary epithelial cells were made quiescent by removal of growth factors, the mRNA for p53 showed a biphasic distribution. p53 mRNA levels were increased during growth arrest, decreased during the G1 phase, and rose again concomitant with the entry of cells into S phase. The immunostaining pattern of p53 also showed a biphasic distribution similar to the pattern of mRNA expression. Despite an increase in p53 mRNA and immunostaining levels, growth factor-arrested cells actually had less total p53 protein. Upon stimulation to proliferate, p53 protein levels remained low throughout G1 and increased concomitant with the entry of cells into S phase. Taken together, the results from these studies demonstrate that p53 immunostaining patterns do not correlate with the overall levels of p53 protein at different times during the cell cycle. Therefore, the distinct changes observed in p53 immunostaining patterns are likely due to posttranslational modifications, conformational changes, or interactions of p53 with other cellular proteins during the cell cycle.
在本研究中，我们使用两种独立方法将正常人乳腺上皮细胞同步至细胞周期的不同时间点，比较p53 mRNA和蛋白质的表达模式。用洛伐他汀处理时，细胞停滞在G1期，通过免疫染色检测发现野生型p53水平似乎升高。代谢阻滞解除后，p53染色阳性的细胞核数量在G1中期急剧下降，随着细胞进入S期又再次增加。与免疫染色结果相反，Northern印迹和Western印迹分析显示，洛伐他汀同步化处理的细胞中p53 mRNA和蛋白质水平变化不大。当通过去除生长因子使正常人乳腺上皮细胞静止时，p53的mRNA呈现双相分布。p53 mRNA水平在生长停滞期间升高，在G1期降低，随着细胞进入S期再次升高。p53的免疫染色模式也显示出与mRNA表达模式相似的双相分布。尽管p53 mRNA和免疫染色水平升高，但生长因子阻滞的细胞中p53总蛋白实际上较少。在刺激增殖后，p53蛋白水平在整个G1期保持较低，随着细胞进入S期而升高。综上所述，这些研究结果表明，p53免疫染色模式与细胞周期不同时间的p53蛋白总体水平不相关。因此，在p53免疫染色模式中观察到的明显变化可能是由于细胞周期中p53的翻译后修饰、构象变化或与其他细胞蛋白的相互作用。
