Gómez Lahoz E, Liegeois N J, Zhang P, Engelman J A, Horner J, Silverman A, Burde R, Roussel M F, Sherr C J, Elledge S J, DePinho R A
Department of Microbiology and Immunology and Department of Medicine, Albert Einstein College of Medicine, Bronx, New York 10461, USA.
Mol Cell Biol. 1999 Jan;19(1):353-63. doi: 10.1128/MCB.19.1.353.
This study examines in vivo the role and functional interrelationships of components regulating exit from the G1 resting phase into the DNA synthetic (S) phase of the cell cycle. Our approach made use of several key experimental attributes of the developing mouse lens, namely its strong dependence on pRb in maintenance of the postmitotic state, the down-regulation of cyclins D and E and up-regulation of the p57(KIP2) inhibitor in the postmitotic lens fiber cell compartment, and the ability to target transgene expression to this compartment. These attributes provide an ideal in vivo context in which to examine the consequences of forced cyclin expression and/or of loss of p57(KIP2) inhibitor function in a cellular compartment that permits an accurate quantitation of cellular proliferation and apoptosis rates in situ. Here, we demonstrate that, despite substantial overlap in cyclin transgene expression levels, D-type and E cyclins exhibited clear functional differences in promoting entry into S phase. In general, forced expression of the D-type cyclins was more efficient than cyclin E in driving lens fiber cells into S phase. In the case of cyclins D1 and D2, ectopic proliferation required their enhanced nuclear localization through CDK4 coexpression. High nuclear levels of cyclin E and CDK2, while not sufficient to promote efficient exit from G1, did act synergistically with ectopic cyclin D/CDK4. The functional differences between D-type and E cyclins was most evident in the p57(KIP2)-deficient lens wherein cyclin D overexpression induced a rate of proliferation equivalent to that of the pRb null lens, while overexpression of cyclin E did not increase the rate of proliferation over that induced by the loss of p57(KIP2) function. These in vivo analyses provide strong biological support for the prevailing view that the antecedent actions of cyclin D/CDK4 act cooperatively with cyclin E/CDK2 and antagonistically with p57(KIP2) to regulate the G1/S transition in a cell type highly dependent upon pRb.
本研究在体内考察了调控细胞从G1静止期进入细胞周期DNA合成（S）期的各组分的作用及其功能相互关系。我们的研究方法利用了发育中小鼠晶状体的几个关键实验特性，即其在维持有丝分裂后状态时对pRb的强烈依赖性、有丝分裂后晶状体纤维细胞区室中细胞周期蛋白D和E的下调以及p57（KIP2）抑制剂的上调，以及将转基因表达靶向至该区室的能力。这些特性提供了一个理想的体内环境，可用于考察在一个能够原位准确量化细胞增殖和凋亡率的细胞区室中，强制表达细胞周期蛋白和/或丧失p57（KIP2）抑制剂功能的后果。在此，我们证明，尽管细胞周期蛋白转基因表达水平存在大量重叠，但D型和E型细胞周期蛋白在促进进入S期方面表现出明显的功能差异。一般来说，在驱动晶状体纤维细胞进入S期方面，强制表达D型细胞周期蛋白比细胞周期蛋白E更有效。就细胞周期蛋白D1和D2而言，异位增殖需要通过共表达CDK4增强其核定位。细胞周期蛋白E和CDK2的高核水平虽然不足以促进从G1期有效退出，但确实与异位细胞周期蛋白D/CDK4协同作用。D型和E型细胞周期蛋白之间的功能差异在p57（KIP2）缺陷型晶状体中最为明显，其中细胞周期蛋白D过表达诱导的增殖速率与pRb缺失型晶状体相当，而细胞周期蛋白E过表达并未使增殖速率超过因p57（KIP2）功能丧失所诱导的增殖速率。这些体内分析为当前的观点提供了有力的生物学支持，即细胞周期蛋白D/CDK4的前期作用与细胞周期蛋白E/CDK2协同作用，并与p57（KIP2）拮抗，以调控高度依赖pRb的细胞类型中的G1/S转换。
