Probably no image is more telling of the defect in the BRCA2
Tr/Tr cell lines than the mitotic chromosome spreads showing multiple breaks and alterations, presumably due to repair defects and mitotic recombination events . Such events are likely to trigger the observed elevations in p53, and may also promote mitotic checkpoint genes, which ensure proper chromosome alignment and segregation.
Lee et al  tested this notion by expressing dominant-negative versions of p53 and Bub1 (N-Bub1) in the BRCA2
Tr/Tr fibroblasts. Significantly, two mutant p53 alleles and a dominant-negative Bub1 construct rescued the proliferation defect in the BRCA2Tr/Tr cells, presumably by suppressing various checkpoints that monitor abnormal chromosome structure or segregation. The authors therefore expected that these growth-rescued cells would now be unleashed to allow even more aberrantly structured chromosomes. The N-Bub1 and the p53R273L expressing cells, although ane-uploid, were surprisingly devoid of abnormal chromosomes. This was not the case for cells that expressed the other p53 allele, G154V, which also rescued the growth defect, but cells retained the abnormal chromosome structures. These intriguing results indicate several mechanisms by which mutations in checkpoint genes could cooperate with BRCA2 mutations to promote aggressive cell growth.
The continued growth of BRCA2
Tr/Tr cells that express the p53 G154V mutant may be more easy to grasp as p53 acts to sense DNA damage and monitor repair, and its absence via mutation or suppression is common to many transformed cells. The apparent lack of chromosomal abnormalities in the cells expressing p53R273L or N-Bub1 is more difficult to comprehend. It is known, for instance, in the absence of p53 or by suppressing Bub1 activity via dominant-negative Bub1, cells avoid cell cycle arrest and apoptosis normally associated with the absence of chromosome alignment in mitosis [7,8]. Whereas these actions might explain the rescue of the growth inhibition of the BRCA2
Tr/Tr cells by p53R273L or N-Bub1, they fail to illuminate why the rescued cells lack chromosomal abnormalities.
Several possible mechanisms are certainly under consideration by researchers. One would have to posit that the abnormal chromosome structures observed in the BRCA2
Tr/Tr cells are in fact reflections of failed repair processes, and that the assembly of the repair complexes onto chromosomes is somehow dependent on particular cell cycle arrest states mediated by p53 or components of the mitotic checkpoint. It would follow that these 'rescued' cells have as much DNA damage but appear normal due to the lack of repair intermediates. The second possibility is that the p53R273L and N-Bub1 mutants allow cells to bypass critical apoptotic events and therefore create a large population of cells from which those with the least chromosomal abnormalities are selected.
Although undoubtedly an area of intense research interest, this conundrum led Lee et al  to examine the thymic lymphomas arising in the BRCA2
Tr/Tr mice for chromosomal abnormalities. Remarkably, cells from these tumors were free of significant chromosomal defects, suggesting a similar loss in checkpoint activities as seen in the BRCA2
Tr/Tr fibroblasts that express p53R273L or N-Bub1. Analysis of the small sample of tumors available (only 1 in 100 of the BRCA2
Tr/Tr mice survive to adulthood) revealed that all had defects in p53, Bub1, or the Bub1-related gene Mad3L. Curiously, three of the lymphomas showed a similar spectrum of genetic defects involving unusual heterozygous deletions of a region around amino acids 140-148 of the p53 gene, as well as identical, in-frame deletion/insertions in the Bub1 gene substituting M290 with codons encoding isoleucine and arginine. A fourth lymphoma showed a heterozygous mutation in the Mad3L gene and lacked obvious changes in p53 or Bub1. Interestingly, all of the p53, Bub1, and Mad3L mutants functioned as dominant-negative agents in cells with regard to the mitotic checkpoint, suggesting that the lymphoma cells are defective in these functions despite the presence of a single wild-type gene.
The work of Lee et al  has implications for mechanisms of cell transformation in general and tumorigenesis involving BRCA2 mutations in particular. For one, it is the second indication, following that of Cahill et al , that mutations in mitotic checkpoint regulators participate in the genomic instability thought to promote tumorigenesis. In the latter case, mutations in Bub1 and Mad3L (BubR1) were found in colorectal carcinoma cell lines displaying a particular genomic instability marked by rapid loss and gains of entire chromosomes (chromosome instability) . It should be noted here that analyses of the Bub1 gene in head and neck squamous cell carcinomas, typically aneuploid tumors, failed to detect mutations . Moreover, Cahill et al  found that only several of the colorec-tal carcinoma cell lines displaying the chromosome instability phenotype showed mutations in the Bub1 or Mad3L genes. It is likely that many, yet to be discovered genes play critical roles in the various checkpoint pathways that govern accurate chromosome segregation, and mutations in such genes could contribute to the genetic instability that promotes tumor cell evolution.