Meijer L, Borgne A, Mulner O, Chong J P, Blow J J, Inagaki N, Inagaki M, Delcros J G, Moulinoux J P
CNRS, Station Biologique, Roscoff, France.
Eur J Biochem. 1997 Jan 15;243(1-2):527-36. doi: 10.1111/j.1432-1033.1997.t01-2-00527.x.
Cyclin-dependent kinases (cdk) play an essential role in the intracellular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic screening has recently led to the discovery of specific inhibitors of cyclin-dependent kinases, such as butyrolactone I, flavopiridol and the purine olomoucine. Among a series of C2, N6, N9-substituted adenines tested on purified cdc2/cyclin B, 2-(1-ethyl-2-hydroxyethylamino)-6-benzylamino-9-isopropylpurine (roscovitine) displays high efficiency and high selectivity towards some cyclin-dependent kinases. The kinase specificity of roscovitine was investigated with 25 highly purified kinases (including protein kinase A, G and C isoforms, myosin light-chain kinase, casein kinase 2, insulin receptor tyrosine kinase, c-src, v-abl). Most kinases are not significantly inhibited by roscovitine. cdc2/cyclin B, cdk2/cyclin A, cdk2/cyclin E and cdk5/p35 only are substantially inhibited (IC50 values of 0.65, 0.7, 0.7 and 0.2 microM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC50 > 100 microM). Extracellular regulated kinases erk1 and erk2 are inhibited with an IC50 of 34 microM and 14 microM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late prophase. Roscovitine inhibits in vitro M-phase-promoting factor activity and in vitro DNA synthesis in Xenopus egg extracts. It blocks progesterone-induced oocyte maturation of Xenopus oocytes and in vivo phosphorylation of the elongation factor eEF-1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC50 of 16 microM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2. In vivo phosphorylation of vimentin on Ser55 by cdc2/cyclin B is inhibited by roscovitine. Through its unique selectivity for some cyclin-dependent kinases, roscovitine provides a useful antimitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.
细胞周期蛋白依赖性激酶（cdk）在细胞分裂周期（cdc）的细胞内调控中起着至关重要的作用。这些激酶及其调节因子在人类肿瘤中经常失调。酶筛选最近导致发现了细胞周期蛋白依赖性激酶的特异性抑制剂，如丁内酯I、黄酮哌啶醇和嘌呤olomoucine。在对纯化的cdc2/细胞周期蛋白B进行测试的一系列C2、N6、N9取代的腺嘌呤中，2-(1-乙基-2-羟乙基氨基)-6-苄基氨基-9-异丙基嘌呤（roscovitine）对某些细胞周期蛋白依赖性激酶表现出高效率和高选择性。用25种高度纯化的激酶（包括蛋白激酶A、G和C同工型、肌球蛋白轻链激酶、酪蛋白激酶2、胰岛素受体酪氨酸激酶、c-src、v-abl）研究了roscovitine的激酶特异性。大多数激酶不受roscovitine的显著抑制。只有cdc2/细胞周期蛋白B、cdk2/细胞周期蛋白A、cdk2/细胞周期蛋白E和cdk5/p35受到显著抑制（IC50值分别为0.65、0.7、0.7和0.2 microM）。roscovitine对cdk4/细胞周期蛋白D1和cdk6/细胞周期蛋白D2的抑制作用非常弱（IC50>；100 microM）。细胞外调节激酶erk1和erk2的IC50分别为34 microM和14 microM。Roscovitine使海星卵母细胞和海胆胚胎在前期晚期可逆性停滞。Roscovitine抑制非洲爪蟾卵提取物中的体外M期促进因子活性和体外DNA合成。它阻断孕酮诱导的非洲爪蟾卵母细胞成熟以及延伸因子eEF-1的体内磷酸化。Roscovitine抑制哺乳动物细胞系的增殖，平均IC50为16 microM。在roscovitine存在下，L1210细胞停滞在G1期并在G2期积累。Roscovitine抑制cdc2/细胞周期蛋白B对波形蛋白Ser55的体内磷酸化。通过对某些细胞周期蛋白依赖性激酶具有独特的选择性，roscovitine为细胞周期研究提供了一种有用的抗有丝分裂试剂，并且可能被证明对于控制具有失调的cdc2、cdk2或cdk5激酶活性的细胞很有意义。
