Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells.
Removal of Cdk inhibitors through both sequestration and downregulation in zearalenone-treated MCF-7 breast cancer cells.
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通过玉米赤霉烯酮处理的 MCF-7 乳腺癌细胞中的隔离和下调来去除 Cdk 抑制剂。
DOI：
10.1002/mc.10048
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发表时间：
2002
期刊：
影响因子：
--
通讯作者：
Wimalasena,Jay
中科院分区：
文献类型：
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作者：
Ahamed,Shamila;Foster,JamesS;Bukovsky,Antonin;Diehl,JAlan;Wimalasena,Jay
真菌雌激素玉米赤霉烯酮作用于MCF-7细胞后，9-12h内可短暂诱导细胞周期蛋白E相关激酶活性，总细胞周期蛋白依赖性蛋白激酶(CDK)2活性在24小时及以后持续升高。细胞周期蛋白E/CDK2活性的增加与CDK抑制因子p27CDK抑制因子1B(P27KIP1)被新形成的细胞周期蛋白D1/CDK4复合体截留和p27KIP1表达下调有关。细胞周期蛋白A/CDK2活性的激活与p27KIP1的实质消除相对应。重组p27KIP1在体外可抑制玉米赤霉烯酮处理的裂解产物中细胞周期蛋白E/CDK2复合体的活性，而重组细胞周期蛋白D1/CDK4复合体的加入可解除这种抑制作用。因此，细胞周期蛋白D1/CDK4对p27KIP1的封闭导致了CDK2在体外的激活。玉米赤霉烯酮处理的细胞裂解产物中的CDK抑制活性被抗p27KIP1和抗CDc2相互作用蛋白(P21CIP1)抗体耗尽。CDK4p16INK4A的CDK4/6特异性CDK抑制剂的过度表达与p27KIP1与CDK2的结合增加有关，并伴随着D细胞周期蛋白/CDK4复合体的破坏。蛋白酶体抑制剂2-亮氨酸-H醛(MG-132)在抑制细胞周期蛋白E/CDK2最初的封闭依赖性激活方面相对无效，但在抑制G1期细胞周期蛋白A/CDK2的后期激活方面与p16INK4A一样有效。玉米赤霉烯酮作用于p16INK4A表达的细胞后，p27KIP1表达下调，延长玉米赤霉烯酮处理时间可逆转p16INK4A表达所致的G1期停滞。玉米赤霉烯酮处理的MCF-7细胞诱导F-box蛋白S相蛋白相关蛋白2(P45SKP2)的表达，这是泛素-连接酶复合体的底物特异性成分，靶向p27KIP1在蛋白酶体中降解。这些研究表明，细胞周期蛋白D1/CDK4复合体对CDK抑制剂的截留和p27KIP1的下调在雌激素诱导MCF-7细胞CDK2活性和S时相进入中起主要作用。©2002 Wiley-Liss公司
Treatment of MCF 7 cells with the fungal estrogen zearalenone induced cyclin E–associated kinase activity transiently within 9–12 h; total cyclin‐dependent kinase (Cdk) 2 activity was elevated for 24 h and beyond. This increased cyclin E/Cdk2 activity was associated with sequestration of the Cdk inhibitor p27 Cdk inhibitor 1B (p27KIP1) by newly formed cyclin D1/Cdk4 complexes and with downregulation of p27KIP1expression. The activation of cyclin A/Cdk2 activity corresponded with virtual elimination of p27KIP1. The activity of cyclin E/Cdk2 complexes from zearalenone‐treated lysates was inhibited in vitro by recombinant p27KIP1, and this inhibition was relieved by the addition of recombinant cyclin D1/Cdk4 complexes. Thus, sequestration of p27KIP1by cyclin D1/Cdk4 resulted in activation of Cdk2 in vitro. Cdk inhibitory activity in lysates of zearalenone‐treated cells was depleted by anti‐p27KIP1and anti‐Cdc2 interacting protein (p21CIP1) antibodies. Overexpression of the Cdk4/6‐specific Cdk inhibitor of Cdk4 p16INK4Awas associated with increased association of p27KIP1with Cdk2, concomitant with disruption of D cyclin/Cdk4 complexes. The proteasome inhibitor 2‐leu‐leu‐leu‐H aldehyde (MG‐132) was relatively ineffective in inhibiting the initial, sequestration‐dependent activation of cyclin E/Cdk2 yet was as effective as p16INK4Ain inhibiting activation of cyclin A/Cdk2 later in G1. Downregulation of p27KIP1proceeded in p16INK4A‐expressing cells after zearalenone treatment, and G1arrest afforded by p16INK4Aexpression was reversible upon prolonged treatment with zearalenone. Zearalenone treatment of MCF‐7 cells elicited expression of F‐box protein S phase kinase–associated protein 2 (p45SKP2), a substrate‐specific component of the ubiquitin‐ligase complex that targets p27KIP1for degradation in the proteasome. These studies suggest that both sequestration of Cdk inhibitors by cyclin D1/Cdk4 complexes and downregulation of p27KIP1play major roles in the induction of Cdk2 activity and S phase entry elicited by estrogens in MCF‐7 cells. © 2002 Wiley‐Liss, Inc.