Prall O W, Sarcevic B, Musgrove E A, Watts C K, Sutherland R L
Cancer Research Program, Garvan Institute of Medical Research, St. Vincent's Hospital, Sydney, New South Wales 2010, Australia.
J Biol Chem. 1997 Apr 18;272(16):10882-94. doi: 10.1074/jbc.272.16.10882.
Estrogens induce cell proliferation in target tissues by stimulating progression through G1 phase of the cell cycle, but the underlying molecular targets remain undefined. To determine the role of the cyclin/cyclin-dependent kinase (CDK)/retinoblastoma protein (pRB) pathway in this response we treated MCF-7 breast cancer cells with the pure estrogen antagonist ICI 182780 to inhibit estrogen-induced gene expression and induce G1 phase arrest. Subsequent treatment with 17beta-estradiol resulted in the synchronous entry of cells into S phase commencing at 12 h. The proportion of cells in S phase reached a maximum of 60% at 21-24 h. Cells subsequently completed mitosis and entered a second semisynchronous round of replication. Entry into S phase was preceded by increased activity of both Cdk4 and cyclin E-Cdk2 and hyperphosphorylation of pRB, all within the first 3-6 h of estradiol treatment. The increase in Cdk4 activity was accompanied by increases in cyclin D1 mRNA and protein, indicating that an initiating event in the activation of Cdk4 was increased cyclin D1 gene expression. In contrast, the levels of Cdk2 and the CDK inhibitors p21 (WAF1/CIP1/SDI1) and p27 (KIP1) in total cell lysates and in cyclin E immunoprecipitates were unaltered at these early time points. However, an inhibitory activity was present in antiestrogen-pretreated cell lysates toward recombinant cyclin E-Cdk2 and was relieved by estradiol treatment. This activity was attributable predominantly to p21. These apparently conflicting data were resolved by performing gel filtration chromatography, which revealed that only a minority of cyclin E-Cdk2 complexes were active following estradiol treatment. Active complexes eluted at a higher molecular weight than inactive complexes, were relatively deficient in both p21 and p27, and contained Cdk2 with increased threonine 160 phosphorylation, consistent with a mechanism of activation of cyclin E-Cdk2 involving both reduced CDK inhibitor association and CDK-activating kinase-mediated phosphorylation of Cdk2. These results provide an explanation for the early activation of both cyclin D1-Cdk4 and cyclin E-Cdk2 complexes that accompany G1-S phase progression in response to estradiol.
雌激素通过刺激细胞周期G1期进程来诱导靶组织中的细胞增殖，但其潜在的分子靶点仍不明确。为了确定细胞周期蛋白/细胞周期蛋白依赖性激酶（CDK）/视网膜母细胞瘤蛋白（pRB）通路在此反应中的作用，我们用纯雌激素拮抗剂ICI 182780处理MCF-7乳腺癌细胞，以抑制雌激素诱导的基因表达并诱导G1期停滞。随后用17β-雌二醇处理导致细胞在12小时开始同步进入S期。S期细胞比例在21 - 24小时达到最大值60%。细胞随后完成有丝分裂并进入第二轮半同步复制。在雌二醇处理的前3 - 6小时内，进入S期之前Cdk4和细胞周期蛋白E - Cdk2的活性均增加，且pRB发生超磷酸化。Cdk4活性的增加伴随着细胞周期蛋白D1 mRNA和蛋白水平的增加，表明细胞周期蛋白D1基因表达增加是激活Cdk4的起始事件。相反，在这些早期时间点，总细胞裂解物和细胞周期蛋白E免疫沉淀中的Cdk2以及CDK抑制剂p21（WAF1/CIP1/SDI1）和p27（KIP1）水平未改变。然而，抗雌激素预处理的细胞裂解物中存在针对重组细胞周期蛋白E - Cdk2的抑制活性，且雌二醇处理可使其解除。这种活性主要归因于p21。通过进行凝胶过滤色谱法解决了这些明显相互矛盾的数据，该方法显示雌二醇处理后只有少数细胞周期蛋白E - Cdk2复合物具有活性。活性复合物比无活性复合物在更高的分子量处洗脱，p21和p27相对较少，并且含有苏氨酸160磷酸化增加的Cdk2，这与细胞周期蛋白E - Cdk2的激活机制一致，该机制涉及CDK抑制剂结合减少以及CDK激活激酶介导的Cdk2磷酸化。这些结果为响应雌二醇的G1 - S期进程中细胞周期蛋白D1 - Cdk4和细胞周期蛋白E - Cdk2复合物的早期激活提供了解释。
