Roscovitine

A4573 cells are injected s.c. into the right posterior flank of CD1 nu/nu mice.

(R)-2-(6-(benzylamino)-9-isopropyl-9H-purin-2-ylamino)butan-1-ol

0.01 - 100 uM

<=50 mg/kg

0.65 uM, 0.65 uM, 0.65 uM, 0.65 uM, 0.65 uM, 0.65 uM

Roscovitine, at a dose of 50 mg/kg, significantly inhibits growth of The Ewing's sarcoma family of tumors (ESFT) xenografts. [4], Roscovitine enhances the antitumor effect of doxorubicin without increased toxicity with a mechanism that involves cell cycle arrest rather than apoptosis in nude mice bearing established MCF7 xenografts. [5]

Enzymes, Kinases activities are assayed at 30 C in buffer C. Blank values are subtracted from the data and activities calculated as molar amount of phosphate incorporated in protein acceptor during a 10-minute incubation. Controls are performed with appropriate dilutions of DMSO. In a few cases, phosphorylation of the substrate is assessed by autoradiography after SDS/PAGE. p34cdc2/cyclin B is purified from M-phase starfish (M. glacialis) oocytes by affinity chromatography. It is assayed with 1 mg histone Hl/mL, in the presence of 15 uM [gamma-32P]ATP (3000 Ci/mmol, 1 mCi/mL) in a final volume of 30 uL. After a 10-minute incubation at 30 C, 25-uL aliquots of supernatant are spotted onto pieces of Whatman P81 phosphocellulose paper, and, after 20 seconds, the filters are washed five times (for at least 5 minutes each time) in a solution of 10mL phosphoric acid/L water. The wet filters are transferred into 6-mL plastic scintillation vials, 5 mL ACS scintillation fluid is added and the radioactivity measured in a Packard counter. The kinase activity is expressed as molar amount of phosphate incorporated in histone H1 during a 10-minutes incubation or as a percentage of maximal activity. p33cdk2/cyclin A and p33cdk2/cyclinE are reconstituted from extracts of sf9 insect cells infected with various baculoviruses. Cyclins A and E are fusion proteins with glutathione S-transferase and the complexes are purified on glutathione-agarose beads. Kinase activities are assayed with 1 mg/mL histone H1, in the presence of 15 uM [gamma-32P]ATP, during 10 minutes, in a final volume of 30 uL, as described for the p34cdc2/cyclin B kinase. p33cdk5/p35 is purified from bovine brain, excluding the Mono S-chromatographic step. The active fractions from the Superose 12 column are pooled and concentrated to a final concentration of approximately 25 ug enzyme/mL. The kinase is assayed with 1 mg/mL histone HI in the presence of 15 uM [gamma-32P]ATP, during 10 minutes in a final volume of 30 uL, as described for the, p34cdc2/cyclin B kinase. p33cdk5/cyclin D1 is obtained from insect cell lysates. Cdk4 is a fusion protein with glutathione-S-transferase and the active complex is purified on glutathione-agarose beads. Its kinase activity is assayed with purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 uM [gamma-32P]ATP, in a final volume of 30 uL. After a 15-minute incubation, 30 uL Laemmli sample buffer is added. The phosphorylated substrate is resolved by 10 % SDS/PAGE and analysed by autoradiography by overnight exposure to Hyperfilm MP and densitometry. p33cdk4/cyclinD 2 is obtained from insect cell lysates. It is assayed with purified retinoblastoma protein (complexed with glutathione-S-transferase) in the presence of 15 uM [gamma-32P]ATP in a final volume of 30 uL. After a 30-minute incubation, 30 uL Laemmli sample buffer is added. The phosphorylated substrate is resolved by 10% SDS/PAGE and analysed by autoradiography by overnight exposure to Hyperfilm MP and densitometry. MAP kinase erkl (tagged with glutathione-S-transferase), is expressed in bacteria, purified on glutathione-agarose beads and assayed with 1 mg myelin basic protein/ml in the presence of 15 uM [gamma-32P]ATP as described above for the p34cdc2cyclin B kinase. His-tagged erkl and erk2 are activated in vitro by mitogen-activated protein kinase kinase, purified (Ni-affinity and Mono Q) and assayed as described above during 10 minutes in a final volume of 30 uL. Protein kinase C isoforms are purified from baculovirus infected sf9 insect cells and assayed with 1 mg/mL protamine sulfate in the presence of 15 uM [gamma-32P]ATP, during 10 minutes at 30 C, in a final volume of 30 uL. Phosphorylated protamine sulfate is recovered on Whatman P81 phosphocellulose paper as described for the cdc2 kinase. The catalytic subunit of cAMP-dependent protein kinase, purified from bovine heart, is assayed with 1 mg histone Hl/ml, in the presence of 15 uM [gamma-32P]ATP as described for the p34cdc2/cyclin B kinase. cGMP-dependent protein kinase, purified to homogeneity from bovine tracheal smooth muscle, is assayed with 1 mg histone Hl/mL, in the presence of 15 uM [gamma-32P]ATP as described for the p34cdc2/cyclin B kinase. Casein kinase 2 is isolated from rat liver cytosol and assayed with 1 mg casein/mL and 15 uM [gamma-32P]ATP. The substrate is spotted on Whatmann 3MM filters and washed with 10% (mass/vol.) trichloroacetic acid. Myosin light chain kinase, purified from chicken gizzard is assayed in the presence of 100 nM calmodulin, 100 uM CaCl2, 50 mM Hepes, 5 mM MgCI, , 1 mM dithiothreitol and 0.1 mg BSA/ml at pH 7.5 using a synthetic peptide based on the smooth-muscle myosin light-chain phosphorylation site and in the presence of 15 uM [gamma-32P]ATP, in a final volume of 50 uL. Incorporation of radioactive phosphate is monitored on phosphocellulose filters as described above. ASK-gamma, a plant homologue of GSK-3, is expressed as a glutathione-S-transferase fusion protein in Escherichia coli and purified on glutathione-agarose. ASK-gamma kinase is assayed, for 10 minutes at 30 C, with 5 ug myelin basic protein, in the presence of 15 uM [gamma-32P]ATP in a final volume of 30 uL. The phosphorylated myelin basic protein is recovered on Whatman P81 phosphocellulose paper as described for the p34cdc2/cyclin B kinase. Insulin receptor tyrosine kinase domain (CIRK-41) is overexpressed in a baculovirus system and purified to homogeneity. Its kinase activity is assayed, for 10 minutes at 30 C, with 5 ug Raytide, in the presence of 15 uM [gamma-32P]ATP, in a final volume of 30 uL. The phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper as described for the p34cdc2/cyclin B kinase. c-src kinase is purified from infected Sf9 cells. The v-abl kinase is expressed in E. coli and affinity purified on IgG Affigel 10. Both kinases are assayed for 10 minutes at 30 C, with 5 ug Raytide, in the presence of 15 uM [gamma-32P]ATP, in a final volume of 30 uL. The phosphorylated Raytide is recovered on Whatman P81 phosphocellulose paper as described for the p34cdc2/cyclin B kinase.

354, 45

Data from [PNAS, 2011, 108, 8417], Roscovitine (Seliciclib, CYC202) purchased from Selleck, In vivo action of R-roscovitine inmouse corticotroph adenomas. Athymic nude mice were s.c. inoculated with corticotroph tumor AtT20 cells (1, 105 cells). Three days after injection, mice were randomized to receive Rroscovitine (150 mg/kg) or vehicle by oral gavage twice daily, 5 d/wk. After 3 wk, tumor xenografts were dissected and (A) tumor volumes were decreased in R-roscovitine-treated animals. (B) Western blot of representative tumor specimens showed decreased ACTH and PCNA expression in R-roscovitine-treated tumors. (C) R-roscovitine-treated corticotroph tumors exhibited decreased PCNA and ACTH coexpressing cells. Fluorescence microscopy image of immunohistochemistry detecting PCNA (red) and ACTH (green) expression in control (a-c) and R-roscovitine-treated tumors (d-f). Cryosection slides were counterstained with DAPI (blue). (D) Blood was collected from each animal for measurement of plasma ACTH and serum corticosterone levels (mean SE, n = 13-14 mice for each group, **P, 0.01).

DMSO 71 mg/mL, Water <1 mg/mL, Ethanol 6 mg/mL