A "spindle assembly" checkpoint has been described that arrests cells in G1 following inappropriate exit from mitosis in the presence of microtubule inhibitors. We have here addressed the question of whether the resulting tetraploid state itself, rather than failure of spindle function or induction of spindle damage, acts as a checkpoint to arrest cells in G1. Dihydrocytochalasin B induces cleavage failure in cells where spindle function and chromatid segregation are both normal. Notably, we show here that nontransformed REF-52 cells arrest indefinitely in tetraploid G1 following cleavage failure. The spindle assembly checkpoint and the tetraploidization checkpoint that we describe here are likely to be equivalent. Both involve arrest in G1 with inactive cdk2 kinase, hypophosphorylated retinoblastoma protein, and elevated levels of p21 WAF1 and cyclin E. Furthermore, both require p53. We show that failure to arrest in G1 following tetraploidization rapidly results in aneuploidy. Similar tetraploid G1 arrest results have been obtained with mouse NIH3T3 and human IMR-90 cells. Thus, we propose that a general checkpoint control acts in G1 to recognize tetraploid cells and induce their arrest and thereby prevents the propagation of errors of late mitosis and the generation of aneuploidy. As such, the tetraploidy checkpoint may be a critical activity of p53 in its role of ensuring genomic integrity. INTRODUCTIONAneuploidy is common among tumor cells and frequently follows after an intermediate tetraploid state (Shackney et al., 1989;Galipeau et al., 1996). The presence of aneuploidy in tumors is correlated with metastatic progression and poor prognosis (Sandberg, 1977;Rabinovitch et al., 1989). A carefully studied progression toward tumor status in Barrett's esophagus cells has shown that the precancerous state is characterized by loss of p53 function, followed by tetraploidy, and then aneuploidy (Galipeau et al., 1996). Subversion of the capacity of the cell to evade the consequences of tetraploidization, which inevitably leads to aneuploidy, may be a common intermediate in the multistep process by which tumor cells arise in situ.Tetraploidy can arise by exit of a cell from mitosis following failures of spindle assembly, of chromosome segregation, or of cytokinesis . Mammalian cells have a checkpoint that maintains cdc2-cyclin B activity and induces mitotic arrest in the presence of inhibitors of microtubule assembly (Kung et al., 1990;Andreassen and Margolis, 1994). This mechanism can logically be expected to ensure the assembly of a functioning mitotic spindle before exit from mitosis. But in actuality, cells have a widely varied capacity to arrest in mitosis in the presence of microtubule inhibitors (Kung et al., 1990;Schimke et al., 1991;Cahill et al., 1998), and many nontransformed cells undergo only a transient mitotic arrest, and then exit mitosis without chromosome segregation and become tetraploid (Minn et al., 1996;Lanni and Jacks, 1998). However, a p53-dependent backup mechanism induces G1 arrest in cell...

We showed in this study that cells deficient of the BRCA1-associated BACH1 helicase, also known as BRIP1, failed to elicit homologous recombination (HR) after DNA double-stranded breaks (DSBs). BACH1-deficient cells were also sensitive to mitomycin C (MMC) and underwent MMC-induced chromosome instability. Moreover, we identified a homozygous nonsense mutation in BACH1 in a FA-J patient-derived cell line and could not detect BACH1 protein in this cell line. Expression of wild-type BACH1 in this cell line reduced the accumulation of cells at G2/M phases following exposure to DNA crosslinkers, a characteristic of Fanconi anemia (FA) cells. These results support the conclusion that BACH1 is FANCJ.

Fanconi anemia (FA) is a human autosomal recessive cancer susceptibility disorder characterized by cellular sensitivity to mitomycin C and defective cell-cycle progression. Six FA genes (corresponding to subtypes A, C, D2, E, F, and G) have been cloned, and the encoded FA proteins interact in a common pathway. DNA damage activates this pathway, leading to monoubiquitination of the downstream FANCD2 protein and targeting to nuclear foci containing BRCA1. In the current study, we demonstrate that FANCD2 also undergoes monoubiquitination during S phase of the cell cycle. Monoubiquitinated FANCD2 colocalizes with BRCA1 and RAD51 in S-phase-specific nuclear foci. Monoubiquitination of FANCD2 is required for normal cell-cycle progression following cellular exposure to mitomycin C. Our data indicate that the monoubiquitination of FANCD2 is highly regulated, and they suggest that FANCD2/BRCA1 complexes and FANCD2/RAD51 complexes participate in an S-phase-specific cellular process, such as DNA repair by homologous recombination. (Blood. 2002; 100:2414-2420)