BIOCHEMICAL AND CELLULAR EFFECTS OF ROSCOVITINE, A POTENT AND SELECTIVE INHIBITOR OF THE CYCLIN-DEPENDENT KINASES CDC2, CDK2 AND CDK5

Cyclin-dependent kinases (cdk) play an essential role in the intracell ular control of the cell division cycle (cdc). These kinases and their regulators are frequently deregulated in human tumours. Enzymatic scr eening has recently led to the discovery of specific inhibitors of cyc lin-dependent kinases, such as butyrolactone I, flavopiridol and the p urine olomoucine. Among a series of C2, N-6, N9-substituted adenines t ested on purified cdc2/cyclin B, hydroxyethylamino)-6-benzylamino-9-is opropylpurine (roscovitine) displays high efficiency and high selectiv ity towards some cyclin-dependent kinases. The kinase specificity of r oscovitine 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 kinas es 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 mu M, respectively). cdk4/cycl in D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC5 0 > 100 mu M). Extracellular regulated kinases erk1 and erk2 are inhib ited with an IC50 of 34 mu M and 14 mu M, respectively. Roscovitine re versibly arrests starfish oocytes and sea urchin embryos in late proph ase. Roscovitine inhibits in vitro M-phase-promoting factor activity a nd in vitro DNA synthesis in Xenopus egg extracts. It blocks progester one-induced oocyte maturation of Xenopus oocytes and in vivo phosphory lation of the elongation factor eEF-1. Roscovitine inhibits the prolif eration of mammalian cell lines with an average IC50 of 16 mu M 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 inh ibited by roscovitine. Through its unique selectivity for some cyclin- dependent kinases, roscovitine provides a useful antimitotic reagent f or cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.