The Cyclin-Dependent Kinase Inhibitors Olomoucine and Roscovitine Arrest Human Fibroblasts in G1 Phase by Specific Inhibition of CDK2 Kinase Activity Francesca Alessi, Santina Quarta,* Monica Savio,* Federica Riva, Laura Rossi,† Lucia A. Stivala,* A. Ivana Scovassi,† Laurent Meijer,‡ and Ennio Prosperi 1 Centro di Studio per l’Istochimica del CNR, Pavia, Italy; *Istituto di Patologia Generale, Universita ` di Pavia, Pavia, Italy; †Istituto di Genetica Biochimica ed Evoluzionistica del CNR, Pavia, Italy; and ‡Station Biologique, CNRS, Roscoff, France The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investi- gated in normal human fibroblasts. Effects on the cell cycle were compared with those induced by the kinase inhibitor staurosporine, which arrests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the related compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Following removal of CDK inhibitors, cells resumed cycle progression en- tering S phase with a kinetics faster than staurosporine- treated samples. Cellular levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhibitors. In contrast, staurosporine significantly reduced the levels of these proteins, as determined by immunocytometry and Western blot analysis. Cyclin A was detectable only in some cells remaining in the G2  M compartment of samples treated with CDK inhibitors, but not in samples treated with staurosporine. Significant reduction in the hyperphosphorylated forms of retinoblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in stau- rosporine-treated samples. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cells treated with olomoucine or roscovitine was markedly inhibited. These results suggest that in normal cells, CDK2 kinase activity is the specific target of olomoucine and roscovitine. © 1998 Academic Press Key Words: olomoucine; roscovitine; staurosporine; cyclin-dependent kinases; Rb protein; cyclins. INTRODUCTION Protein kinase complexes formed by the association of cyclins and their catalytic subunits called cyclin- dependent kinases (CDKs) play an important role in cell cycle progression [1–3]. Regulation of the activity of these complexes in normal and tumor cells has been extensively investigated [4, 5]. However, the identity of the protein substrates of these kinases is far from being completely discovered [6]. The use of chemical inhibitors specific for CDKs could provide further in- formation on the key points of cell cycle progression. In addition, such a class of molecules may represent a novel tool for inhibition of tumor cell growth. The pu- rine derivative olomoucine has proved to be a potent and specific inhibitor of CDKs, in particular of the purified cyclin B/CDK1, cyclin A/CDK2, and cyclin E/CDK2 complexes, in in vitro assays [7, 8]. Olo- moucine was shown to affect the growth of a large variety of plant and animal cells. In fact, this com- pound was found to inhibit the G1/S transition of uni- cellular algae, to arrest mesophyl protoplasts in G1 phase, and to delay the maturation of starfish, Xeno- pus, and mouse oocytes [9, 10]. The growth of various tumor cell lines was inhibited at the G1 and S phase transit [10], and induction of a G1 and/or G2 arrest was observed [11, 12]. At the molecular level, a direct effect on DNA synthesis was observed in vitro, in Xenopus extracts [7]. In addition, both CDK1 and CDK2 kinases immunoprecipitated from CTLL-2 cells were inhibited in vitro by olomoucine, thus suggesting an explanation for the growth arrest observed at the G1/S and G2/M boundaries [10]. However, direct evidence of the in vivo target specificity of olomoucine is not yet available and, at the molecular level, the effects induced by purine derivatives on cell cycle progression are only partially understood [13]. A number of proteins relevant for cell cycle control may be considered in order to investigate the specificity of kinase inhibitors and to locate the time of their effects along the cell cycle. The proliferating cell nu- clear antigen (PCNA), the cofactor of DNA polymerase  [14, 15], has been shown to bind tightly to DNA replication sites during S phase, by forming a complex 1 To whom reprint requests should be addressed at Centro di Studio per l’Istochimica del CNR, Piazza Botta 10, 27100 Pavia, Italy. Fax: +(39) 382 506430. E-mail: prosperi@dragon.ian.pv.cnr.it. 8 0014-4827/98 $25.00 Copyright © 1998 by Academic Press All rights of reproduction in any form reserved. EXPERIMENTAL CELL RESEARCH 245, 8 –18 (1998) ARTICLE NO. EX984216