Inhibiting BCR-ABL kinase activity up-regulates p27 and down-regulates SKP2. Mo7e cells expressing p210^BCR-ABL were treated with imatinib (2.5 μM) for 16 hours. (A) Immunoblot analysis of whole-cell lysates with antiphosphotyrosine antibodies. (B) The cell-cycle profile was analyzed by propidium iodide (PI) staining. Mo7e cells treated with imatinib (2.5 μM) for 16 hours were used as a control. (C) Immunoblot analysis of the effect of BCR-ABL kinase inhibition on p27, cyclin E, CDK2, pT187p27, SKP2, CKS1, KPC1, KPC2 expression levels in nuclear (N) and cytoplasmic extracts (C). Sp1 and α-tubulin were used to assess the purity of the nuclear and cytoplasmic fractions, respectively, and actin was used as a loading control. No effect on p27 and SKP2 expression was observed in Mo7e cells treated with imatinib under similar conditions. (D) Analysis of p27 in nuclear (N) and cytoplasmic (C) lysates of Mo7e p210^BCR-ABL cells shows accumulation of p27 predominantly in the nucleus compared with the cytoplasm. Densitometry was performed to quantitate p27 levels from 3 independent experiments. All densitometry values are given in Boehringer light units (BLU). (E) Inhibition of BCR-ABL kinase activity with imatinib reduces CDK2 activity toward histone H1. Mo7e cells expressing p210^BCR-ABL were treated with 2.5 μM imatinib for 16 hours and lysates from whole cells as well as from nuclear and cytoplasmic fractions were immunoprecipitated with an antibody against CDK2. Immunoprecipitates were incubated in kinase buffer in the presence of [γ³²P]-ATP and histone H1 as a substrate. CDK2 kinase activity toward histone H1 was measured by scintillation counting in 3 independent experiments. Error bars represent SD.