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 ade‐nines tested on purified cdc2/cylin B, 2‐(1‐ethyl‐2‐hydroxyethylamino)‐6‐benzylamino‐9‐isopropylpu‐rine (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 cdkSlp35 only are substantially inhibited (IC₅₀values of 0.65, 0.7, 0.7 and 0.2 μM, respectively). cdk4/cyclin D1 and cdk6/cyclin D2 are very poorly inhibited by roscovitine (IC₅₀>100 μM). Extracellular regulated kinases erkl and erk2 are inhibited with an IC₅₀of 34 μM and 14 μM, respectively. Roscovitine reversibly arrests starfish oocytes and sea urchin embryos in late pro‐phase. Roscovitine inhibitsin vitroM‐phase‐promoting factor activity andin vitroDNA synthesis inXenopusegg extracts. It blocks progesterone‐induced oocyte maturation ofXenopusoocytes andin vivophosphorylation of the elongation factor eEF‐1. Roscovitine inhibits the proliferation of mammalian cell lines with an average IC₅₀] of 16 μM. In the presence of roscovitine L1210 cells arrest in G1 and accumulate in G2.In vivophosphorylation 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 anti‐mitotic reagent for cell cycle studies and may prove interesting to control cells with deregulated cdc2, cdk2 or cdk5 kinase activities.

In late mitosis and early G1, Mcm2–7 complexes are loaded onto DNA to license replication origins for use in the upcoming S phase. However, the amount of Mcm2–7 loaded is in significant excess over the number of origins normally used. We show here that in human cells, excess chromatin-bound Mcm2–7 license dormant replication origins that do not fire during normal DNA replication, in part due to checkpoint activity. Dormant origins were activated within active replicon clusters if replication fork progression was inhibited, despite the activation of S-phase checkpoints. After lowering levels of chromatin-bound Mcm2–7 in human cells by RNA interference (RNAi), the use of dormant origins was suppressed in response to replicative stress. Although cells with lowered chromatin-bound Mcm2–7 replicated at normal rates, when challenged with replication inhibitors they had dramatically reduced rates of DNA synthesis and reduced viability. These results suggest that the use of dormant origins licensed by excess Mcm2–7 is a new and physiologically important mechanism that cells utilize to maintain DNA replication rates under conditions of replicative stress. We propose that checkpoint kinase activity can preferentially suppress initiation within inactive replicon clusters, thereby directing new initiation events toward active clusters that are experiencing replication problems.

While testing purines related to the non‐specific protein kinase inhibitors N⁶‐dimethylaminopurine and N⁶‐(²‐isopentenyl)adenine as potential inhibitors of the p34^cdc2/cyclin B kinase, we discovered a compound with high specificity, 2‐(2‐hydroxyethylamino)‐6‐benzylamino‐9‐methylpurine (olomoucine). Kinetic analysis of kinase inhibition reveals that olomoucine behaves as a competitive inhibitor for ATP and as a non‐competitive inhibitor for histone H1 (linear inhibition for both substrates). The kinase specificity of this inhibition was investigated for 35 highly purified kinases (including p34^cdk4/cyclin D1, p40^cdk6/cyclin D3, CAMP‐dependent and cGMP‐dependent kinases, eight protein kinase C isoforms, calmodulin‐dependent kinase II, myosin light‐chain kinase, mitogen‐activated S6 kinase, casein kinase 2, double‐stranded RNA‐activated protein kinase, AMP‐stimulated kinase, eight tyrosine kinases). Most kinases are not significantly inhibited. Only the cell‐cycle regulating p34^cdc2/cyclin B, p33^cdk2/cyclin A and p33^cdk2/cyclin E kinases, the brain p33^cdk5/p35 kinase and the ERK1/MAP‐kinase (and its starfish homologue p44^mpk) are substantially inhibited by olomoucine (IC₅₀ values are 7, 7, 7, 3 and 25 μM, respectively). The cdk4/cyclin D1 and cdk6/cyclin D3 kinases are not significantly sensitive to olomoucine (IC₅₀ values greater than 1 mM and 150 μM, respectively). N⁶‐(²‐Isopentenyl)adenine is confirmed as a general kinase inhibitor with IC₅₀, values of 50–100 μM for many kinases. The purine specificity of cyclin‐dependent kinase inhibition was investigated: among 81 purine derivatives tested, only C2, N6 and N9‐substituted purines exert a strong inhibitory effect on the p34^cdc2/cyclin B kinase. An essentially similar sensitivity to this olomoucine family of compounds was observed for the brain‐specific cdk5/p35 kinase. Structure/activity relationship studies allow speculation on the interactions of olomoucine and its analogues with the kinase catalytic subunit. Olomoucine inhibits in vitro M‐phase‐promoting factor activity in metaphase‐arrested Xenopus egg extracts, inhibits in vitro DNA synthesis in Xenopus interphase egg extracts and inhibits the licensing factor, an essential replication factor ensuring that DNA is replicated only once in each cell cycle. Olomoucine inhibits the starfish oocyte G2/M transition in vivo. Through its unique selectivity olomoucine provides an anti‐mitotic reagent that may preferentially inhibit certain steps of the cell cycle.