CHFR binds to and regulates MAD2 in the spindle checkpoint through its cysteine-rich domain

CHFR has been implicated as a tumor suppressor in a multitude of cancers. It was originally identified as a major component of the antephase checkpoint. Recently, CHFR was reported to interact with MAD2, an important component of the spindle assembly checkpoint, where CHFR knockdown resulted in mislocalization of MAD2 and disruption of the MAD2/CDC20 interaction. To further understand how CHFR interacts with MAD2, we deleted key functional domains of CHFR, and investigated the effect on MAD2 binding and function. Here we show that deletion of the cysteine-rich domain of CHFR is required for the CHFR/MAD2 interaction as well as proper localization of MAD2 in the cell. Furthermore, the cysteine-rich domain deletion exhibits impaired ability to promote the MAD2/CDC20 interaction, leading to an increase in mitotic defects relative to wild type CHFR. These data support a critical role for CHFR in the MAD2 spindle checkpoint. Furthermore, these data establish the cysteine-rich domain of CHFR as the essential domain for the CHFR/MAD2 interaction and for promoting interaction between MAD2 and CDC20 to inhibit the anaphase-promoting complex.     

Polyploidization increases the sensitivity to DNA-damaging agents in mammalian cells

Polyploidization occurs during normal development as well as during tumorigenesis. In this study, we investigated if the responses to genotoxic stress in cancer cells are influenced by the ploidy. Prolonged treatment of Hep3B cells with the spindle inhibitor nocodazole resulted in mitotic slippage, followed by re-replication of the DNA to produce polyploids. Reintroduction of p53 restored the checkpoints and suppressed polyploidization. Remarkably, a stable tetraploidy cell line could be generated from Hep3B by a transient nocodazole treatment followed by a period of recovery. Using this novel tetraploid system, we found that tetraploidization increased the cell volume without significantly affecting the cell cycle. Although tetraploidization was accompanied by an increase in centrosome number, the majority of mitoses in the tetraploid cells remained bipolar. Polyploidization sensitized cells to genotoxic stress inflicted by ionizing radiation and topoisomerase inhibitors without affecting the sensitivity to spindle inhibitors. Accordingly, more gamma-H2AX foci were induced by radiation in tetraploids than in normal Hep3B cells. Likewise, primary tetraploid human fibroblasts displayed higher gamma-H2AX foci formation than diploid human fibroblasts. An implication for chemotherapy is that some cancer cells can be sensitized to genotoxic agents by a preceding step that induces polyploidization.     

Chromator is required for proper microtubule spindle formation and mitosis in Drosophila

The chromodomain protein, Chromator, has been shown to have multiple functions that include regulation of chromatin structure as well as coordination of muscle remodeling during metamorphosis depending on the developmental context. In this study we show that mitotic neuroblasts from brain squash preparations from larvae heteroallelic for the two Chromator loss-of-function alleles Chro⁷¹ and Chro⁶¹² have severe microtubule spindle and chromosome segregation defects that were associated with a reduction in brain size. The microtubule spindles formed were incomplete, unfocused, and/or without clear spindle poles and at anaphase chromosomes were lagging and scattered. Time-lapse analysis of mitosis in S2 cells depleted of Chromator by RNAi treatment suggested that the lagging and scattered chromosome phenotypes were caused by incomplete alignment of chromosomes at the metaphase plate, possibly due to a defective spindle-assembly checkpoint, as well as of frayed and unstable microtubule spindles during anaphase. Expression of full-length Chromator transgenes under endogenous promoter control restored both microtubule spindle morphology as well as brain size strongly indicating that the observed mutant defects were directly attributable to lack of Chromator function.