Figure 6

Inhibition of CDK2 kinase activity and induction of apoptosis by small molecule inhibitors of p27 depletions (SMIPs). (a) Histone H1 kinase assays were performed with CDK2 complexes immunopurified from LNCaP-S14 cells treated with 40 μM SMIPs for 24 h as described in Methods. Roscovitine (R) was used as positive control. (b) Histone H1 kinase assays with CDK2 complexes immunopurified from dimethyl sulfoxide (DMSO) treated LNCaP-S14 cells. The indicated SMIPs (20 μM) were added to the reactions. Roscovitine (R) was used as a positive control for kinase inhibition in vitro. (c) Analysis of CDK2 complexes. Equal amounts of protein from LNCaP-S14 cells treated with DMSO, SMIPs (40 μM) or roscovitine (R, 20 μM) were immunoprecipitated using an anti-CDK2 antibody as described in the Methods section. The levels of co-precipitated cyclin E, cyclin A, p27, and p21 were evaluated by immunoblotting. (d) LNCaP-S14 cells were transfected with siRNAs for p27 and p21 as described in the Methods section and treated with DMSO or SMIPs (40 μM) for 24 h. The cell cycle distribution was analysed by flow cytometry. The graphs represent the population of SMIPs-treated cells in G1 and G2 normalized to DMSO-treated cells for each siRNA. (e) Total cell lysates from siRNA transfected cells were subjected to immunoblotting with the specified antibodies. The blots are representative of three independent experiments. (f) Apoptosis was evaluated in total cell lysates from LNCaP-S14 cells treated with 40 μM SMIPs for 24 h. Poly ADP ribosome polymerase (PARP) cleavage was visualized using an antibody that detects both full length (Fl) and cleaved (Cl) PARP. The blot is representative of three independent experiments. (g) Quantification of mono and oligonucleosomes as indication of apoptosis. Lysates of cells treated with DMSO or SMIPs (40 μM) for 24 h were obtained and analysed for cytoplasmic histone-associated DNA fragments as described in the Methods section. The graph represents the DNA fragmentation enrichment factor ± standard deviation of two independent experiments.