Identification of a new murine tumor necrosis factor receptor locus that contains two novel murine receptors for tumor necrosis factor-related apoptosis-inducing ligand (TRAIL).

Tumor necrosis factor (TNF) ligand and receptor superfamily members play critical roles in diverse developmental and pathological settings. In search for novel TNF superfamily members, we identified a murine chromosomal locus that contains three new TNF receptor-related genes. Sequence alignments suggest that the ligand binding regions of these murine TNF receptor homologues, mTNFRH1, -2 and -3, are most homologous to those of the tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) receptors. By using a number of in vitro ligand-receptor binding assays, we demonstrate that mTNFRH1 and -2, but not mTNFRH3, bind murine TRAIL, suggesting that they are indeed TRAIL receptors. This notion is further supported by our demonstration that both mTNFRH1:Fc and mTNFRH2:Fc fusion proteins inhibited mTRAIL-induced apoptosis of Jurkat cells. Unlike the only other known murine TRAIL receptor mTRAILR2, however, neither mTNFRH2 nor mTNFRH3 has a cytoplasmic region containing the well characterized death domain motif. Coupled with our observation that overexpression of mTNFRH1 and -2 in 293T cells neither induces apoptosis nor triggers NFkappaB activation, we propose that the mTnfrh1 and mTnfrh2 genes encode the first described murine decoy receptors for TRAIL, and we renamed them mDcTrailr1 and -r2, respectively. Interestingly, the overall sequence structures of mDcTRAILR1 and -R2 are quite distinct from those of the known human decoy TRAIL receptors, suggesting that the presence of TRAIL decoy receptors represents a more recent evolutionary event

Targeting the Mitotic Checkpoint to Kill Tumor Cells

One of the most common hallmarks of cancer cells is aneuploidy or an abnormal number of chromosomes. This abnormal chromosome content is a consequence of chromosome missegregation during mitosis, a defect that is seen more frequently in tumor cell divisions as in normal cell divisions. In fact, a large fraction of human tumors display a chromosome instable phenotype, meaning that they very frequently missegregate chromosomes. This can cause variegated aneuploidy within the tumor tissue. It has been argued that this hallmark of cancer could be exploited in anti-cancer therapies. Here we test this hypothesis by inactivation of the mitotic checkpoint through RNAi-mediated depletion of an essential checkpoint component, Mps1. The mitotic checkpoint delays segregation of chromosomes during mitosis until all chromosomes are properly attached to the mitotic spindle. Its inactivation will therefore lead to increased segregation errors. Indeed, we show that this can lead to increased cell death in tumor cells. We demonstrate that increased cell death is associated with a dramatic increase in segregation errors. This suggests that inhibition of the mitotic checkpoint might represent a useful anti-cancer strategy

Caspase-2-mediated cell death is required for deleting aneuploid cells

Caspase-2, one of the most evolutionarily conserved of the caspase family, has been implicated in maintenance of chromosomal stability and tumour suppression. Caspase-2 deficient (Casp2-/-) mice develop normally but show premature ageing-related traits and when challenged by certain stressors, succumb to enhanced tumour development and aneuploidy. To test how caspase-2 protects against chromosomal instability, we utilized an ex vivo system for aneuploidy where primary splenocytes from Casp2-/- mice were exposed to anti-mitotic drugs and followed up by live cell imaging. Our data show that caspase-2 is required for deleting mitotically aberrant cells. Acute silencing of caspase-2 in cultured human cells recapitulated these results. We further generated Casp2C320S mutant mice to demonstrate that caspase-2 catalytic activity is essential for its function in limiting aneuploidy. Our results provide direct evidence that the apoptotic activity of caspase-2 is necessary for deleting cells with mitotic aberrations to limit aneuploidy.S Dawar, Y Lim, J Puccini, M White, P Thomas, L Bouchier-Hayes, D R Green, L Dorstyn and S Kuma

APC15 drives the turnover of MCC-CDC20 to make the spindle assembly checkpoint responsive to kinetochore attachment

Faithful chromosome segregation during mitosis depends on the Spindle Assembly Checkpoint (SAC) that monitors kinetochore attachment to the mitotic spindle. Unattached kinetochores generate mitotic checkpoint proteins complexes (MCCs) that bind and inhibit the Anaphase Promoting Complex/Cyclosome (APC/C). How the SAC proficiently inhibits the APC/C but still allows its rapid activation when the last kinetochore attaches to the spindle is important to understand how cells maintain genomic stability. We show that the APC/C subunit APC15 is required for the turnover of the APC/C co-activator Cdc20 and release of MCCs during SAC signalling but not for APC/C activity per se. In the absence of APC15, MCCs and ubiquitylated Cdc20 remain ‘locked’ onto the APC/C, which prevents the ubiquitylation and degradation of Cyclin B1 when the SAC is satisfied. We conclude that APC15 mediates the constant turnover of Cdc20 and MCCs on the APC/C to allow the SAC to respond to the attachment state of kinetochores

Ran-dependent docking of importin-β to RanBP2/Nup358 filaments is essential for protein import and cell viability

RanBP2 captures RanGTP–importin-β complexes at cytoplasmic fibrils to ensure adequate classical NLS–mediated protein import and cell viability

Spindle Assembly Checkpoint Regulates Mitotic Cell Cycle Progression during Preimplantation Embryo Development

Errors in chromosome segregation or distribution may result in aneuploid embryo formation, which causes implantation failure, spontaneous abortion, genetic diseases, or embryo death. Embryonic aneuploidy occurs when chromosome aberrations are present in gametes or early embryos. To date, it is still unclear whether the spindle assembly checkpoint (SAC) is required for the regulation of mitotic cell cycle progression to ensure mitotic fidelity during preimplantation development. In this study, using overexpression and RNA interference (RNAi) approaches, we analyzed the role of SAC components (Bub3, BubR1 and Mad2) in mouse preimplantation embryos. Our data showed that overexpressed SAC components inhibited metaphase-anaphase transition by preventing sister chromatid segregation. Deletion of SAC components by RNAi accelerated the metaphase-anaphase transition during the first cleavage and caused micronuclei formation, chromosome misalignment and aneuploidy, which caused decreased implantation and delayed development. Furthermore, in the presence of the spindle-depolymerizing drug nocodazole, SAC depleted embryos failed to arrest at metaphase. Our results suggest that SAC is essential for the regulation of mitotic cell cycle progression in cleavage stage mouse embryos

Mutation of the Zebrafish Nucleoporin elys Sensitizes Tissue Progenitors to Replication Stress

The recessive lethal mutation flotte lotte (flo) disrupts development of the zebrafish digestive system and other tissues. We show that flo encodes the ortholog of Mel-28/Elys, a highly conserved gene that has been shown to be required for nuclear integrity in worms and nuclear pore complex (NPC) assembly in amphibian and mammalian cells. Maternal elys expression sustains zebrafish flo mutants to larval stages when cells in proliferative tissues that lack nuclear pores undergo cell cycle arrest and apoptosis. p53 mutation rescues apoptosis in the flo retina and optic tectum, but not in the intestine, where the checkpoint kinase Chk2 is activated. Chk2 inhibition and replication stress induced by DNA synthesis inhibitors were lethal to flo larvae. By contrast, flo mutants were not sensitized to agents that cause DNA double strand breaks, thus showing that loss of Elys disrupts responses to selected replication inhibitors. Elys binds Mcm2-7 complexes derived from Xenopus egg extracts. Mutation of elys reduced chromatin binding of Mcm2, but not binding of Mcm3 or Mcm4 in the flo intestine. These in vivo data indicate a role for Elys in Mcm2-chromatin interactions. Furthermore, they support a recently proposed model in which replication origins licensed by excess Mcm2-7 are required for the survival of human cells exposed to replication stress