The Cyclin-Dependent Kinase Inhibitors Olomoucine and Roscovitine Arrest Human Fibroblasts in G1 Phase by Specific Inhibition of CDK2 Kinase Activity

doi:10.1006/excr.1998.4216

Experimental Cell Research

Volume 245, Issue 1, 25 November 1998, Pages 8-18

https://doi.org/10.1006/excr.1998.4216 Get rights and content

Abstract

The specificity and the temporal location of cell cycle arrest induced by the cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated 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 theirin vitroactivity, 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 entering 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 staurosporine-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.

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^☆: L. Meijer, S. Guidet

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:[email protected].

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Regular ArticleThe Cyclin-Dependent Kinase Inhibitors Olomoucine and Roscovitine Arrest Human Fibroblasts in G1 Phase by Specific Inhibition of CDK2 Kinase Activity☆

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Regular Article
The Cyclin-Dependent Kinase Inhibitors Olomoucine and Roscovitine Arrest Human Fibroblasts in G1 Phase by Specific Inhibition of CDK2 Kinase Activity☆