Recombinant cyclin/CDK holoenzymes are purified from Sf9 cells engineered to produce baculoviruses that express a specific cyclin or CDK. Cyclin/CDK complexes are typically diluted to a final concentration of 50 μg/mL in a kinase reaction buffer containing 50 mM Tris-HCl (pH 8.0), 10 mM MgCl₂, 1 mM DTT, and 0.1 mM sodium orthovanadate. For each kinase reaction, 1 μg of enzyme and 20 μL of a 2 μM substrate solution (a biotinylated peptide derived from histone H1) are mixed and combined with 10 μL of diluted Dinaciclib (SCH 727965). The reaction is started by the addition of 50 μL of 2 μM ATP and 0.1 μCi of ³³P-ATP. Kinase reactions are incubated for 1 hour at room temperature and are stopped by the addition of 0.1% Triton X-100, 1 mM ATP, 5 mM EDTA, and 5 mg/mL streptavidin-coated SPA beads. SPA beads are captured using a 96-well GF/B filter plate and a Filtermate universal harvester. Beads are washed twice with 2 M NaCl and twice with 2 M NaCl containing 1% phosphoric acid. The signal is then assayed using a TopCount 96-well liquid scintillation counter. Dose-response curves are generated from duplicate, eight-point serial dilutions of inhibitory compounds. IC₅₀ values are derived by nonlinear regression analysis^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

A2780 cells are plated onto tissue culture dishes and propagated with the appropriate growth media. Growing cultures are exposed to increasing concentrations of Dinaciclib (0.75, 1.5, 3.15, 6.25, 12.5, 25, and 500 nM) or a vehicle control, typically for 7 days. After removing the medium, cells are fixed with 50% methanol/50% acetone for 5 minutes and stained with 0.2% crystal violet in 2% ethanol for 5 minutes. Following staining, cells are washed with 5 to 10 mL of water. Stained cells are solubilized in 1% deoxycholic acid, and the absorbance of the resulting solution is measured at 600 nm using a SOFTmax PRO 4.3 plate reader. Absorbance of Dinaciclib-treated samples is plotted as a percent of that of a vehicle-treated control, and data are reported as an IC₅₀ value relative to these controls. For suspension cell lines, assessments of cell viability are obtained using the alamarBlue Cell Viability Assay kit^[1].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

Mice^[1]
For tumor implantation, specific cell lines are grown in vitro, washed once with PBS, and resuspended in 50% Matrigel in PBS to a final concentration of 4×10⁷ to 5×10⁷ cells per milliliter. Nude mice are injected with 0.1 mL of this suspension s.c. in the flank region. Tumor length (L), width (W), and height (H) are measured by a caliper twice weekly on each mouse and then used to calculate tumor volume using the formula (L×W×H)/2. When the tumor volume reaches 100 mm³, the animals are randomized to treatment groups (10 mice/group) and treated i.p. with either Dinaciclib (8, 16, 32, and 48 mg/kg daily, i.p.) or individual chemotherapeutic agents according to the dosing schedule indicated in table and figure legends. Tumor volumes and body weights are measured during and after the treatment periods.

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Parry D, et al. Dinaciclib (SCH 727965), a novel and potent cyclin-dependent kinase inhibitor. Mol Cancer Ther. 2010 Aug;9(8):2344-53.  [Content Brief]

  [2]. Feldmann G, et al. Cyclin-dependent kinase inhibitor Dinaciclib (SCH727965) inhibits pancreatic cancer growth and progression in murine xenograft models. Cancer Biol Ther. 2011 Oct 1;12(7):598-609.  [Content Brief]