The Aurora/Ipl1 family of protein kinases plays multiple roles in mitosis and cytokinesis. Here, we describe ZM447439, a novel selective Aurora kinase inhibitor. Cells treated with ZM447439 progress through interphase, enter mitosis normally, and assemble bipolar spindles. However, chromosome alignment, segregation, and cytokinesis all fail. Despite the presence of maloriented chromosomes, ZM447439-treated cells exit mitosis with normal kinetics, indicating that the spindle checkpoint is compromised. Indeed, ZM447439 prevents mitotic arrest after exposure to paclitaxel. RNA interference experiments suggest that these phenotypes are due to inhibition of Aurora B, not Aurora A or some other kinase. In the absence of Aurora B function, kinetochore localization of the spindle checkpoint components BubR1, Mad2, and Cenp-E is diminished. Furthermore, inhibition of Aurora B kinase activity prevents the rebinding of BubR1 to metaphase kinetochores after a reduction in centromeric tension. Aurora B kinase activity is also required for phosphorylation of BubR1 on entry into mitosis. Finally, we show that BubR1 is not only required for spindle checkpoint function, but is also required for chromosome alignment. Together, these results suggest that by targeting checkpoint proteins to kinetochores, Aurora B couples chromosome alignment with anaphase onset.

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During mitosis, the recruitment of spindle-checkpoint-associated proteins to the kinetochore occurs in a defined order. The protein kinase Bub1 localizes to the kinetochore very early during mitosis, followed by Cenp-F, BubR1, Cenp-E and finally Mad2. Using RNA interference, we have investigated whether this order of binding reflects a level of dependency in human somatic cells. Specifically, we show that Bub1 plays a key role in the assembly of checkpoint proteins at the kinetochore, being required for the subsequent localization of Cenp-F, BubR1, Cenp-E and Mad2. In contrast to studies in Xenopus, we also show that BubR1 is not required for kinetochore localization of Bub1. Repression of Bub1 increases the number of cells with lagging chromosomes at metaphase, suggesting that Bub1 plays a role in chromosome congression. However, repression of Bub1 does not appear to compromise spindle checkpoint function either during normal mitosis or in response to spindle damage. This raises the possibility that, in the absence of Bub1, other mechanisms contribute to spindle checkpoint function.

APC is often cited as a prime example of a tumor suppressor gene. Truncating germline and somatic mutations (or, infrequently, allelic loss) occur in tumors in FAP (familial adenomatous polyposis). Most sporadic colorectal cancers also have two APC mutations. Clues from attenuated polyposis, missense germline variants with mild disease and the somatic mutation cluster region (codons 1,250-1,450) indicate, however, that APC mutations might not result in simple loss of protein function. We have found that FAP patients with germline APC mutations within a small region (codons 1,194-1,392 at most) mainly show allelic loss in their colorectal adenomas, in contrast to other FAP patients, whose 'second hits' tend to occur by truncating mutations in the mutation cluster region. Our results indicate that different APC mutations provide cells with different selective advantages, with mutations close to codon 1,300 providing the greatest advantage. Allelic loss is selected strongly in cells with one mutation near codon 1,300. A different germline-somatic APC mutation association exists in FAP desmoids. APC is not, therefore, a classical tumor suppressor. Our findings also indicate a new mechanism for disease severity: if a broader spectrum of mutations is selected in tumors, the somatic mutation rate is effectively higher and more tumors grow.