Li Y, Jenkins C W, Nichols M A, Xiong Y
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 27599.
Oncogene. 1994 Aug;9(8):2261-8.
In normal human fibroblast cells, the primary cell cycle regulators, the cyclin-dependent kinases (CDKs), exist predominantly in multiple quaternary complexes, each consisting of a CDK, a cyclin, proliferating cell nuclear antigen (PCNA) and p21. p21 encodes a universal inhibitor of cyclin-dependent kinases. Here we show that the level of p21 mRNA and the interaction of p21 protein with cyclin-CDK enzymes are regulated during the cell cycle. When normal human fibroblast IMR90 cells were released from serum starvation, p21 mRNA reached its highest level immediately following serum stimulation, began to decrease at the G1/S boundary, fell to its lowest level during S phase, and accumulated again as cells exited from S phase. p21 protein associates with each cyclin-CDK complex in a cell cycle dependent manner. Cyclin A-CDK2-p21-PCNA and Cyclin B1-CDC2-p21-PCNA complexes are assembled in early S and G2 phase, respectively, indicating that p21 and/or PCNA regulates the enzymatic activity of each kinase at the time of their functioning. Cyclin D1-CDK4-p21-PCNA complexes, on the other hand, persist throughout the cell cycle, suggesting that cyclin D1-CDK4 quaternary complexes may play a role in monitoring an event(s) that may occur at any time, rather than at a specific stage of the cell cycle. The level of p21 mRNA in early passage Li-Fraumeni cells that are heterozygous for p53 mutation remained similar to that in normal fibroblasts, but was undetectable in immortalized Li-Fraumeni cells homozygous for mutant p53. This finding provides a plausible molecular explanation for the loss of genetic stability associated with cells homozygous, but not heterozygous, for p53 mutation.
在正常人类成纤维细胞中，主要的细胞周期调节因子，即细胞周期蛋白依赖性激酶（CDK），主要以多种四级复合物的形式存在，每种复合物都由一个CDK、一个细胞周期蛋白、增殖细胞核抗原（PCNA）和p21组成。p21编码一种细胞周期蛋白依赖性激酶的通用抑制剂。在这里，我们表明p21 mRNA的水平以及p21蛋白与细胞周期蛋白-CDK酶的相互作用在细胞周期中受到调节。当正常人类成纤维细胞IMR90从血清饥饿状态中释放出来时，p21 mRNA在血清刺激后立即达到最高水平，在G1/S边界开始下降，在S期降至最低水平，并在细胞退出S期时再次积累。p21蛋白以细胞周期依赖性方式与每种细胞周期蛋白-CDK复合物结合。细胞周期蛋白A-CDK2-p21-PCNA和细胞周期蛋白B1-CDC2-p21-PCNA复合物分别在S期早期和G2期组装，这表明p21和/或PCNA在其发挥作用时调节每种激酶的酶活性。另一方面，细胞周期蛋白D1-CDK4-p21-PCNA复合物在整个细胞周期中持续存在，这表明细胞周期蛋白D1-CDK4四级复合物可能在监测可能在任何时间发生的事件中发挥作用，而不是在细胞周期的特定阶段。早期传代的携带p53突变杂合子的李-弗劳梅尼细胞中p21 mRNA的水平与正常成纤维细胞中的水平相似，但在携带p53突变纯合子的永生化李-弗劳梅尼细胞中无法检测到。这一发现为与p53突变纯合子而非杂合子细胞相关的遗传稳定性丧失提供了一个合理的分子解释。
