| The maintenance of genomic integrity is central to an organism's survival. This is ensured by several checkpoint mechanisms, the inactivation of which, could contribute to carcinogenesis. chfr, a newly described gene, defines a checkpoint acting at the G2/M boundary. However, little is known about this checkpoint at the biochemical or cellular levels or its relevance to cancer. To clarify the point of Chfr-induced cell cycle arrest, we characterized cells that do not express chfr and stably-transfected derivatives that express wild-type chfr. After exposure to microtubule poisons, chfr expression induced a transient cell cycle arrest delaying entry into prometaphase. The chfr-expressing cells were apparently arrested in early prophase and could readily be distinguished from cells arrested in G2 by DNA damage by virtue of, their nuclear morphology, suggesting partial lamin depolymerization, reorganization of the actin cytoskeleton, and partial Cyclin B1-Cdc2 activity. In addition, neither endogenous Plk1 protein levels nor localization were affected by chfr, in contrast to a recent report. Instead, analysis of Chfr's ubiquitin ligase activity revealed that Chfr can catalyze the formation of non-canonical Lys63-linked polyubiquitin chains with Ubc13-Mms2 acting as the ubiquitin-conjugating enzyme. Ubc13-Mms2 and Lys63-polyubiquitin chains are not associated with targeting proteins to the proteasome, but rather with signaling cellular stress. Thus, Chfr may have a role in signaling the presence of mitotic stress induced by microtubule poisons. Furthermore, analysis of chfr expression and sequence in cell lines and primary tumors have shown that chfr is frequently inactivated by both epigenetic and genetic mechanisms in human cancers. These studies demonstrate the Chfr checkpoint is a bona fide mitotic checkpoint, which utilizes the non-canonical Ubc13/Mms2 ubiquitin pathway. Importantly, this data coupled with the inactivation of Chfr in cancers illustrates for the first time, frequent inactivation of a mitotic checkpoint gene in cancer. |
