The cyclin-dependent kinase inhibitors olomoucine and roscovitine arrest human fibroblasts in G1 phase by specific inhibition of CDK2 kinase activity

The specificity and the temporal location of cell cycle arrest induced by t he cyclin-dependent kinase (CDK) inhibitors olomoucine and roscovitine were investigated in normal human fibroblasts. Effects on the cell cycle were c ompared with those induced by the kinase inhibitor staurosporine, which arr ests normal cells in early G1 phase by acting upstream of CDK2. Consistent with their in vitro activity, olomoucine and roscovitine, but not the relat ed compound iso-olomoucine, induced a dose-dependent arrest in G1 phase. Fo llowing removal of CDK inhibitors, cells resumed cycle progression entering S phase with a kinetics faster than staurosporine-treated samples. Cellula r levels of PCNA, cyclin D1, and cyclin E were not affected by the CDK inhi bitors. In contrast, staurosporine significantly reduced the levels of thes e proteins, as determined by immunocytometry and Western blot analysis. Cyc lin 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 st aurosporine. Significant reduction in the hyperphosphorylated forms of reti noblastoma protein was found in samples treated with CDK inhibitors, while only hypophosphorylated forms were observed in staurosporine-treated sample s. Concomitantly, CDK2, but not CDK4, activity immunoprecipitated from cell s treated with olomoucine or roscovitine was markedly inhibited. These resu lts suggest that in normal cells, CDK2 kinase activity is the specific targ et of olomoucine and roscovitine. (C) 1998 Academic Press.