Cyclin E2 induces genomic instability by mechanisms distinct from cyclin E1

Cyclins E1 drives the initiation of DNA replication, and deregulation of its periodic expression leads to mitotic delay associated with genomic instability. Since it is not known whether the closely related protein cyclin E2 shares these properties, we overexpressed cyclin E2 in breast cancer cells. This did not affect the duration of mitosis, nor did it cause an increase in p107 association with CDK2. In contrast, cyclin E1 overexpression led to inhibition of the APC complex, prolonged metaphase and increased p107 association with CDK2. Despite these different effects on the cell cycle, elevated levels of either cyclin E1 or E2 led to hallmarks of genomic instability, i.e., an increased proportion of abnormal mitoses, micronuclei and chromosomal aberrations. Cyclin E2 induction of genomic instability by a mechanism distinct from cyclin E1 indicates that these two proteins have unique functions in a cancer setting.     

Differences in degradation lead to asynchronous expression of cyclin E1 and cyclin E2 in cancer cells

Cyclin E1 is expressed at the G₁/S phase transition of the cell cycle to drive the initiation of DNA replication and is degraded during S/G₂M. Deregulation of its periodic degradation is observed in cancer and is associated with increased proliferation and genomic instability. We identify that in cancer cells, unlike normal cells, the closely related protein cyclin E2 is expressed predominantly in S phase, concurrent with DNA replication. This occurs at least in part because the ubiquitin ligase component that is responsible for cyclin E1 downregulation in S phase, Fbw7, fails to effectively target cyclin E2 for proteosomal degradation. The distinct cell cycle expression of the two E-type cyclins in cancer cells has implications for their roles in genomic instability and proliferation and may explain their associations with different signatures of disease.     

A cell-cycle-regulated kinase activity phosphorylates plant retinoblastoma protein and contains, in Arabidopsis, a CDKA/cyclin D complex

The activity of cyclin-dependent kinases (CDK) is crucial for cell-cycle transitions. Here, we report the identification of a CDK activity that phosphorylates the retinoblastoma-related (RBR) protein. A CDK/cyclin complex that binds to and phosphorylates RBR may be isolated from various plant sources, e.g. wheat, maize, Arabidopsis thaliana and tobacco, and from cells growing under various conditions. The presence of an RBR-associated CDK activity correlates with the proliferative activity, suggesting that phosphorylation of RBR is a major event in actively proliferating tissues. In A. thaliana, this activity comprises a PSTAIRE CDKA and at least cyclin D2. Furthermore, this CDK activity is cell-cycle-regulated, as revealed by studies with highly synchronized tobacco BY-2 cells where it is maximal in late G1 and early S phase cells and progressively decreases until G2 phase. Aphidicolin-arrested but not roscovitine-arrested cells contain a PSTAIRE-type CDK that binds to and phosphorylates RBR. Thus, association with a D-type cyclin is a likely mechanism leading to CDK activation late in G1. Our studies constitute the first report measuring the activity of CDK/cyclin complexes formed in vivo on RBR, an activity that fluctuates in a cell-cycle-dependent manner. This work provides the basis for further studies on the impact of phosphorylation of RBR on its function during the cell cycle and development.     

过表达E2F6基因抑制BRD7基因启动子活性

BRD7基因是采用cDNA代表性差异分析法克隆的一个新Bromodomain基因(GenBank 登录号AF152604)。它在鼻咽癌细胞和组织中表达明显下调,过表达BRD7基因可抑制鼻咽癌细胞的生长和细胞周期的进程。前期工作已克隆了BRD7基因启动子区,并将其启动子定位于450bp（-404→+46bp）的区域。为了进一步揭示BRD7基因在鼻咽癌细胞和组织中表达下调的分子机制,生物信息学分析表明BRD7启动子区有E2F6转录因子结合位点,电泳迁移率实验结果表明转录因子E2F6特异性地结合于BRD7启动子区。荧光素酶检测和绿色荧光蛋白表达检测都证实过表达E2F6基因能抑制BRD7基因启动子活性     

Increased cyclin E expression may obviate the role of cyclin D1 during brain development in cyclin D1 knockout mice

Cyclins D and E play critical roles during the G1 phase of mammalian cell division. Cyclin D1 expression is high and expected to play an important role during mouse brain development. However, in the present study, we found no difference in CNS morphology between cyclin D1 knockout (KO) and control wild-type mice at the ages of 1, 4 and 12 months. Analysis of protein expression in embryonic brains revealed that cyclin E is obviously increased in cyclin D1 KO mice at 13.5 days post coitum. At the same age a high level of cyclin D1 expression is detected in the embryonic brain of wild-type mice. The data indicate that enhanced cyclin E protein expression in cyclin D1 KO mice may obviate the role of cyclin D1 and contribute to the normal brain development of cyclin D1 KO mice.     

转录因子E2F1抑制CREG表达调控体外培养的人血管平滑肌细胞分化

为探讨转录因子E2F1在血管平滑肌细胞(vascular smooth muscle cells,VSMCs)表型转化中的作用及其对E1A激活基因阻遏子(cellular repressor of E1A-stimulated genes,CREG)表达调控的分子机制,应用生物信息学方法,定位人CREG(hCREG)基因启动子并确定转录因子E2F1在hCREG启动子区的结合位点,PCR方法克隆并构建hCREG基因启动子绿色荧光报告基因载体,以hCREG启动子区E2F1结合位点为模板,化学合成E2F1寡聚脱氧核苷酸(ODN)和错配E2F1ODN,利用转录因子"诱骗(Decoy)"策略,用E2F1ODN转染体外培养的VSMCs以阻断E2F1与hCREG基因启动子区的结合,蛋白质印迹(Western blot)分析检测阻断前后细胞内hCREG蛋白、报告基因绿色荧光蛋白(green fluorescent protein,GFP)和平滑肌细胞分化标志蛋白SMα-actin表达变化.结果显示:分化表型HITASY细胞中E2F1表达下调伴出核转位,而增殖表型的HITASY细胞中E2F1蛋白表达明显增加且定位于核内.进一步应用FuGene6瞬时转染E2F1ODN和错配E2F1ODN于体外培养HITASY细胞中,蛋白质印迹分析发现,转染E2F1ODN后,HITASY细胞中hCREG、SMα-actin和GFP表达均较未阻断组及错配组细胞明显增加.上述研究结果证实,E2F1是hCREG基因转录的重要调控因子,能够直接结合于hCREG启动子区阻遏hCREG表达,参与hCREG蛋白对VSMCs表型转化的调控作用.