title: SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells.
abstract: The E2F family plays a critical role in the expression of genes required for entry into and progression through S phase. E2F-mediated transcription is repressed by the tumor suppressor retinoblastoma protein (pRb), which results in sequestration of E2F in a multiprotein complex that includes pRb. Derepression of E2F results from a series of complex phosphorylation events mediated by cyclin D/cdk4 and cyclin E/cdk2. We have employed a novel 3-substituted indolinone compound, 3-[1-(3H-imidazol-4-yl)-meth-(Z)-ylidene]-5-methoxy-1,3-dihydro-indol-2-one (SU9516), which selectively inhibits cdk2 activity (Lane et al., Cancer Res 2001;61:6170-7) to investigate these events. Electrophoretic mobility gel shift assays were performed on SU9516-treated and -untreated HT-29, SW480, and RKO human colon cancer cell extracts. 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%. These findings delineate a previously undescribed mechanism for SU9516-mediated cell growth arrest through down-regulation of cyclin D1, inhibition of cdk2 levels and activity, and pan-sequestration of E2F.
authors.affiliation: Albert Einstein Comprehensive Cancer Center and the Albert Einstein College of Medicine, Bronx, NY 10463, USA.
authors.forename: Bo
authors.initials: B
authors.lastname: Yu
authors.forename: Maureen E
authors.initials: ME
authors.lastname: Lane
authors.forename: Scott
authors.initials: S
authors.lastname: Wadler
mesh_terms: D042846:CDC2-CDC28 Kinases; D002352:Carrier Proteins / Q000494:pharmacology; D018797:Cell Cycle Proteins; D002478:Cells, Cultured; D003110:Colonic Neoplasms / Q000473:pathology; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016213:Cyclins / Q000378:metabolism; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; D050703:E2F2 Transcription Factor; D050699:E2F3 Transcription Factor; D050700:E2F5 Transcription Factor; D019073:HT29 Cells; D006801:Humans; D007093:Imidazoles / Q000494:pharmacology*; D007211:Indoles / Q000494:pharmacology*; D047908:Intracellular Signaling Peptides and Proteins; D008970:Molecular Weight; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D016160:Retinoblastoma Protein / Q000378:metabolism; D014157:Transcription Factors / Q000378:metabolism*
chemical_list: D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D016213:Cyclins; D004268:DNA-Binding Proteins; D050684:E2F Transcription Factors; D050687:E2F1 Transcription Factor; C493753:E2F1 protein, human; D050703:E2F2 Transcription Factor; D050699:E2F3 Transcription Factor; C098319:E2F3 protein, human; D050700:E2F5 Transcription Factor; C092558:E2F5 protein, human; D007093:Imidazoles; D007211:Indoles; D047908:Intracellular Signaling Peptides and Proteins; D016160:Retinoblastoma Protein; C433985:SU 9516; D014157:Transcription Factors; C015482:protein kinase modulator; D017346:Protein Serine-Threonine Kinases; D042846:CDC2-CDC28 Kinases; C495894:CDK2 protein, human; D051357:Cyclin-Dependent Kinase 2; D018844:Cyclin-Dependent Kinases