In vitro activity: SU9516 is a potent inhibitor for cdk 2 and that it is 9-fold and 1.8-fold more selective for cdk2 than cdk4 and cdk1, respectively. Treatment with SU9516 (5 μM) inhibited (P ≤ 0.05) both cdk2-specific (27–64%) and cdk4-specific (26–49%) phosphorylation of pRb in SW480 cells at all (24, 48, and 72 h) time points. RKO cells remained blocked in G2-M at 20 h post-serum induction and -addition of SU9516. SU9516 produced a dose-dependent G1 accumulation in EGF-stimulated cells. Treatment with 5 microM SU9516 prevented dissociation of pRb from E2F1 in all cell lines (HT-29>RKO>SW480). Treatment effects were time-dependent, demonstrating greater inhibition at 48 hr versus 24hr in HT-29 cells. Furthermore, E2F species were sequestered in complexes with p107, p130, DP-1, and cyclins A and E. After a 24-hr treatment with 5 microM SU9516, cyclin D1 and cdk2 levels decreased by 10-60%. Exposure of U937 and other leukemia cells to SU9516 concentrations> or =5 microM rapidly (i.e., within 4 h) induced cytochrome c release, Bax mitochondrial translocation, and apoptosis in association with pronounced down-regulation of the antiapoptotic protein Mcl-1.

Kinase Assay: Kinase assays were performed in 96-well polypropylene plates. Each reaction contained 2 μg of histone H1 at a final concentration of 10 μM [γ-33P]ATP (0.2 μCi/well; approximately twice the experimentally determined Km; data not shown), 10 mM MgCl2, 1 mM DTT, 0.01% Triton X-100, and 10% glycerol in a 40 μl volume. The reaction was initiated with the addition of 20 μl enzyme (6ng cdk2/well resulting in a final concentration of 1.6 nM), which was previously diluted 1:50-1:200 in the same buffer, and allowed to proceed for 1 h at room temperature. Reaction was stopped by the addition of 0.01 ml 10% phosphoric acid, and 25 μl of reaction mixture was transferred to P30 phosphocellulose filter mat paper. The filter mat was washed three times with 1.0% phosphoric acid, air dried, and then counted for radioactivity in a liquid scintillation counter (Wallac Betaplate). Cell assay(SRB Assay for Cell Proliferation): RKO cells and SW480 cells were seeded in replicates (n = 6) in 96-well plates (Becton Dickinson, Lincoln Park, NJ) at 1 × 104 cells/well and allowed to attach overnight. SU9516 (Sugen, San Francisco, CA) was added in concentrations from 0.05 μM to 50.00 μM for 24 h, the cells were then washed twice with PBS, and cells were replenished with complete media. The cells were fixed at 0, 4, and 7 days post-drug removal and assayed for protein levels using a modified SRB cytotoxicity assay. The cells were fixed in 10% trichloroacetic acid for 1 h, washed in distilled H2O, and stained in 0.4% SRB/acetic acid for 30 min. The cells were then washed in 0.1% acetic acid, solubilized in 10 mM Tris (pH 9), and analyzed on a Bio-Rad 360 microplate reader (Bio-Rad) at 595 nm. All experiments were repeated at least three times.

Cell Assay: In RKO cells, SU 9516 (5 μM) decreased cdk2-specific phosphorylation of pRb by 52%. While, in SW480 cells, SU9516 (5 μM) inhibited both cdk2-specific and cdk4-specific phosphorylation of pRb by 64% and 49%, respectively. Also, SU 9516 (5 μM) resulted in G0-G1 or G2-M block and induced apoptosis in a dose-dependent way. In HT-29, SW480 and RKO human colon cancer cells, SU9516 (5 μM) inhibited dissociation of pRb from E2F1 in a time-dependant way. Also, SU 9516 decreased Cyclin D1 and CDK2 by 10-60%. In human leukemia cells, SU 9516 (5 μM) induced Bax mitochondrial translocation, cytochrome c release and apoptosis, which were associated with down-regulation of the antiapoptotic protein Mcl-1. Also, SU 9516 induced activation of caspase-3 and -8

Cancer Res. 2001 Aug 15;61(16):6170-7; Biochem Pharmacol. 2002 Oct 1;64(7):1091-100.