SU9516, a cyclin-dependent kinase 2 inhibitor, promotes accumulation of high molecular weight E2F complexes in human colon carcinoma cells.

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.