Loss of Emi1-Dependent Anaphase-Promoting Complex/Cyclosome Inhibition Deregulates E2F Target Expression and Elicits DNA Damage-Induced Senescence

Abstract

Expression of the anaphase-promoting complex/cyclosome (APC/C) inhibitor Emi1 is required for the accumulation of APC/C substrates crucial for DNA synthesis and mitotic entry. We show that in vivo Emi1 expression correlates with the proliferative status of the cellular compartment and that cells lacking Emi1 undergo cellular senescence. Emi1 depletion leads to strong decreases in E2F target mRNA and APC/C substrate protein abundances. However, cyclin E mRNA and cyclin E protein levels and associated kinase activities are increased. Cells lacking Emi1 undergo DNA damage, likely explained by replication stress upon deregulated cyclin E- and A-associated kinase activities. Inhibition of ATM kinase prevents induction of senescence, implying that senescence is a consequence of DNA damage. Surprisingly, no senescence or no extensive amount of senescence is evident upon depletion of the Emi1-stabilizing factor Evi5 or Pin1, respectively. Our data suggest that maintenance of a protein stabilization/mRNA expression positive-feedback circuit fueled by Emi1 is required for accurate cell cycle progression, maintenance of DNA integrity, and prevention of cellular senescence.

SUPPLEMENTAL MATERIAL

We thank Rob Vries and Peter Verrijzer for providing C1 SNF5-inducible cells and Mei Hsieh for generating maltose-binding protein-Emi1 immunogen. We also thank the Genentech Inc. microarray facilities, Zora Modrusan and Cynthia Honchell for help with microarray analysis, Matt Brauer for statistical analysis, Jeff Eastham-Anderson for help with image analysis, and the Genentech Inc. oligonucleotide synthesis core for generation of siRNA oligonucleotides. We are grateful to T. Halazonetis for communicating results prior to publication, members of the Jackson lab for insightful discussions, and Jorge Torres, Adam Eldridge, Tom O'Brien, and Andrea Cochran for critical reading of the manuscript.

This work was supported by a Damon Runyon Cancer Research Foundation fellowship (DRG-1811-04) to E.W.V., NIH grant K08 NS45077 to N.L.L., and NIH grants RO1 GM054811 and RO1 GM063023 to P.K.J.