Because many oncoproteins (such as c-MYC, c-JUN, cyclin D, and H-RAS) are known substrates of RBX1-SCF E3 ligases (2,32) and may accumulate upon RBX1 silencing to trigger DDR, we measured the level of these oncoproteins in U87 cells

Because many oncoproteins (such as c-MYC, c-JUN, cyclin D, and H-RAS) are known substrates of RBX1-SCF E3 ligases (2,32) and may accumulate upon RBX1 silencing to trigger DDR, we measured the level of these oncoproteins in U87 cells. == RO-1138452 SCF (Skp1/Cullins/F-box; also known as CRL (Cullin-RINGLigase) E3 ubiquitin ligases are the largest multiunit E3 ligases that promote the degradation of numerous short-lived cellular proteins, including cell cycle regulators, transcription factors, signal transducers, and oncogene/tumor suppressors (1,2). A recent global protein stability profiling analysis identified 350 potential SCF substrates, the majority of which were previously unidentified (3). Most recently, a study Rabbit Polyclonal to ELOVL1 of SCF inactivation by a small molecule inhibitor of cullin neddylation suggested that up to 20% of ubiquitinated cellular proteins are mediated by SCF E3 for proteasome degradation (4). Thus, RO-1138452 SCF E3 ubiquitin ligases regulate many aspects of cellular functions and biological processes under physiological conditions. Dysfunction of SCF is involved in the pathogenesis of a variety of diseases, including cancer (2,5). The core of SCF ubiquitin ligases is a complex of RBX1-cullins (6). RBX1 consists of 108 amino acids with a C-terminal RING-H2 finger domain required for zinc ion binding and ligase activity (79). Crystal structure studies revealed that RBX1 complexes with cullin/F-box proteins form functional SCF E3 ligases that transfer ubiquitin from E2 to specific substrates for proteasome-targeted degradation (10). Previous studies have shown that RBX1 interacts with all seven cullin family members to activate E3 ubiquitin ligases and regulate numerous biological processes by promoting timely degradation of cellular substrates (9,11). As an essential component of SCF E3 ligase, RBX1 plays a critical role in development. In yeast, deletion ofHrt1, the yeast homolog of RBX1, causes lethality, which can be rescued by human RBX1 or RBX2/SAG (Sensitive to Apoptosis gene) (9,12,13). InCaenorhabditis elegans, RBX-1 is crucial for cell cycle progression and chromosome metabolism, and RBX-1 silencing results in embryonic death (14,15). InDrosophila, Roc1a, theDrosophilahomolog of RBX1, is required for cell proliferation and embryonic development, and deletion of Roc1a results in animal death (16). Recently, we reported that mouseRbx1disruption causes early embryonic lethality due to significant accumulation of p27 to suppress proliferation, which can be partially rescued by a simultaneous deletion of p27 (17). These findings suggest that thein vivophysiological function ofRbx1is to ensure cell proliferation during embryonic development. Consistently, RBX1 was found to be essential for cancer cell proliferation (18) and survival (19). It appears that cancer cells are addicted to RBX1-overexpressed environments. Upon RBX1 siRNA silencing, cancer cells sequentially undergo G2-M arrest, senescence, and apoptosis, which are associated with a DNA damage response (DDR)4(19). In this study, we demonstrate that RBX1 silencing actually causes DNA double-strand breaks (DSB), leading to chromosome aneuploidy, which is associated with the accumulation of DNA replication licensing proteins CDT1 and ORC1. We further demonstrate that RBX-1 silencing inC. eleganstriggers similar DDR in intestinal cells, which can be completely rescued by simultaneous silencing of CDT-1. Thus, RBX1-SCF E3 ubiquitin ligases play an essential role in genomic stability in bothin vitrocultured cells andin vivoanimals. == EXPERIMENTAL PROCEDURES == == == == == == Cell Culture == H1299 human lung cancer cells and U87 human glioblastoma RO-1138452 cells were purchased from American Type Culture Collection and grown at 37 C in 5% CO2in DMEM supplemented with 10% FBS. == siRNA Silencing == U87 and H1299 cells were transfected with siRNA oligonucleotides (made by Dharmacon) RO-1138452 using Lipofectamine 2000. The siRNA sequences are as follows: for RBX1, 5-GACTTTCCCTGCTGTTACCTAA-3; for CDT1, 5-CGTGGATGAAGTACCC GAC-3; for ORC1, 5-CTGCACTACCAAACCTATA-3; for CHK1, SMARTpool M-003255-04; for CHK2, SMARTpool.