Trembley J H, Ebbert J O, Kren B T, Steer C J
Department of Medicine, University of Minnesota Medical School, Minneapolis 55455, USA.
Cell Growth Differ. 1996 Jul;7(7):903-16.
Cyclin genes and their products are important regulatory participants in the eukaryotic cell cycle. It is well established that cyclin B1 protein forms a complex with cyclin-dependent kinase 1 (CDK1), which, when activated, initiates mitosis. We have previously established that cyclin B1 gene expression is posttranscriptionally regulated in regenerating rat liver after 70% partial hepatectomy (PH). We now report further characterization of cyclin B1 gene expression, as well as that of CDK1 and cdc25B, in this unique in vivo model of cell proliferation. Cyclin B1 transcripts were detected by RNase protection through 96 h of liver regeneration and exhibited dramatic changes in steady-state levels. Peak expressions occurred at 24-30 h, more significantly at 42-48, and at 72 h. By Northern blot analysis, single transcripts for CDK1 and cdc25B were detected, and the temporal expression of both transcripts during liver regeneration mirrored that of cyclin B1. By Western blot and immunohistochemical analyses, cyclin B1 protein levels did not change significantly in either nuclear or cytoplasmic fractions, whereas CDK1 protein levels paralleled their associated RNA expression. Cdc25B protein levels steadily decreased from 0 to 96 h after PH. In addition, cytoplasmic protein levels of cyclin B1 exhibited a constant distribution in subfractions of microsome- and polysome-associated and free proteins. Cyclin B1 RNA also localized to these three cytoplasmic subfractions. Finally, the apparent translational activity of cyclin B1 transcripts was very similar at both 24 and 48 h after PH, in contrast to their respective mRNA half-lives. In a peroxisome proliferation model of hepatocyte growth and apoptosis, cyclin B1 and CDK1 proteins were induced in the absence of transcript up-regulation. Our results demonstrate that cyclin B1 mRNA steady-state levels are regulated posttranscriptionally in regenerating rat liver. Furthermore, the pattern of cyclin B1 transcript expression is paralleled by that of the CDK1 gene, whereas their respective protein steady-state levels provide a striking contrast. Finally, cyclin B1 is differentially regulated by an uncoupling of transcript abundance and translational processing in two in vivo models of hepatocyte growth. The abundance of cyclin B1 protein in nonreplicating cells suggests that cyclin B1 may be available for other cellular pathways in the hepatocyte.
细胞周期蛋白基因及其产物是真核细胞周期中重要的调节参与者。众所周知，细胞周期蛋白B1与细胞周期蛋白依赖性激酶1（CDK1）形成复合物，该复合物激活后会启动有丝分裂。我们之前已经确定，在70%部分肝切除（PH）后的再生大鼠肝脏中，细胞周期蛋白B1基因的表达受到转录后调控。我们现在报告在这个独特的细胞增殖体内模型中，对细胞周期蛋白B1基因表达以及CDK1和细胞分裂周期蛋白25B（cdc25B）表达的进一步特征分析。通过核糖核酸酶保护法在肝脏再生的96小时内检测到细胞周期蛋白B1转录本，其稳态水平呈现出显著变化。峰值表达出现在24 - 30小时，在42 - 48小时更为显著，以及在72小时。通过Northern印迹分析，检测到CDK1和cdc25B的单条转录本，并且在肝脏再生过程中这两种转录本的时间表达模式与细胞周期蛋白B1的相似。通过蛋白质免疫印迹和免疫组织化学分析，细胞周期蛋白B1的蛋白质水平在核或细胞质组分中均未发生显著变化，而CDK1的蛋白质水平与其相关RNA表达平行。肝切除后0至96小时，cdc25B的蛋白质水平稳步下降。此外，细胞周期蛋白B1的细胞质蛋白质水平在与微粒体和多核糖体相关的亚组分以及游离蛋白质中呈现恒定分布。细胞周期蛋白B1 RNA也定位于这三个细胞质亚组分。最后，与它们各自的mRNA半衰期相反，细胞周期蛋白B1转录本在肝切除后24小时和48小时的表观翻译活性非常相似。在肝细胞生长和凋亡的过氧化物酶体增殖模型中，细胞周期蛋白B1和CDK1蛋白在转录本未上调的情况下被诱导。我们的结果表明，在再生大鼠肝脏中，细胞周期蛋白B1 mRNA的稳态水平受到转录后调控。此外，细胞周期蛋白B1转录本的表达模式与CDK1基因的相似，而它们各自的蛋白质稳态水平形成了显著对比。最后，在两种肝细胞生长的体内模型中，细胞周期蛋白B1通过转录本丰度与翻译加工的解偶联受到不同调控。非复制细胞中细胞周期蛋白B1蛋白的丰度表明，细胞周期蛋白B1可能参与肝细胞中的其他细胞途径。
