Suchergebnisse

The Anaphase-promoting complex/cyclosome (APC/C) cofactor Cdh1 modulates cell proliferation by targeting multiple cell-cycle regulators for ubiquitin-dependent degradation. Lack of Cdh1 results in structural and numerical chromosome aberrations, a hallmark of genomic instability. By using a proteomic approach in Cdh1-null cells and mouse tissues, we have identified kinesin Eg5 and topoisomerase 2α as Cdh1 targets involved in the maintenance of genomic stability. These proteins are ubiquitinated and degraded through specific KEN and D boxes in a Cdh1-dependent manner. Whereas Cdh1-null cells display partial resistance to Eg5 inhibitors such as monastrol, lack of Cdh1 results in a dramatic sensitivity to Top2α poisons as a consequence of increased levels of trapped Top2α-DNA complexes. Chemical inhibition of the APC/C in cancer cells results in increased sensitivity to Top2α poisons. This work identifies in vivo targets of the mammalian APC/C-Cdh1 complex and reveals synthetic lethal interactions of relevance in anticancer treatments. ; We thank Angeles Almeida, Thomas U. Mayer, William T. Beck, Anthony A. Hyman, Jan-Michael Peters, Eusebio Manchado, and Scott Lowe for reagents. M. E., A.J.L.-C., and M.A.-F. were supported by the Spanish Ministry of Education, Culture and Sports, the AECC Scientific Foundation, and the EU-PEOPLE programme, respectively. Work in the O.F.-C. laboratory was supported by grants from the Spanish Ministry of Economy and Competitiveness (MINECO; SAF2011-23753), the Association for International Cancer Research (120229), the Howard Hughes Medical Institute, and the European Research Council (ERC-210520). Work in the H.Y. laboratory was funded by grants from Marie Curie Cancer Care and Cancer Research UK. Work in M.M.'s laboratory was funded by grants from the Foundation Ramon Areces, MINECO (SAF2012-38215), the OncoCycle Programme (S2010/BMD-2470) from the Comunidad de Madrid, and the European Union Seventh Framework Programme (MitoSys project; HEALTH-F5-2010-241548). ; Sí

Reprogramming is a dynamic process that can result in multiple pluripotent cell types emerging from divergent paths. Cell surface protein expression is a particularly desirable tool to categorize reprogramming and pluripotency as it enables robust quantification and enrichment of live cells. Here we use cell surface proteomics to interrogate mouse cell reprogramming dynamics and discover CD24 as a marker that tracks the emergence of reprogramming-responsive cells, while enabling the analysis and enrichment of transgene-dependent (F-class) and -independent (traditional) induced pluripotent stem cells (iPSCs) at later stages. Furthermore, CD24 can be used to delineate epiblast stem cells (EpiSCs) from embryonic stem cells (ESCs) in mouse pluripotent culture. Importantly, regulated CD24 expression is conserved in human pluripotent stem cells (PSCs), tracking the conversion of human ESCs to more naive-like PSC states. Thus, CD24 is a conserved marker for tracking divergent states in both reprogramming and standard pluripotent culture. ; We thank Kento Onishi for providing EpiSC samples. We also thank Dr Jeff Wrana and Dr Dan Trcka for providing secondary DOX-inducible reprogramming system to allow further validation of our findings. We would also like to acknowledge the assistance and support of laboratory colleagues and collaborators. This work has been supported by a GL2 grant from Ontario Government (AN) and a CIHR Grant to P.W.Z. N.S. is the recipient of the NSERC Vanier Canada Graduate Scholarship. A.N. is Tier 1 Canada Research Chair in Stem Cells and Regeneration. P.W.Z. is the Canada Research Chair in Stem Cell Bioengineering. ; Sí