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A high degree of aneuploidy characterizes the majority of human tumors. Aneuploid status can arise through mitotic or cleavage failure coupled with failure of tetraploid G(1) checkpoint control, or through deregulation of centrosome number, thus altering the number of mitotic spindle poles. p53 and the RB pocket proteins are important to the control of G(1) progression, and p53 has previously been suggested as important to the control of centrosome duplication. We demonstrate here that neither suppression of p53 nor of the RB pocket protein family directly generates altered centrosome numbers in any of several mammalian primary cell lines. Instead, amplification of centrosome number occurs in two steps. The first step is failure to arrest at a G(1) tetraploidy checkpoint after failure to segregate the genome in mitosis, and the second step is clustering of centrosomes at a single spindle pole in subsequent tetraploid or aneuploid mitosis. The trigger for these events is mitotic or cleavage failure that is independent of p53 or RB status. Finally, we find that mouse embryo fibroblasts spontaneously enter tetraploid G(1), explaining the previous demonstration of centrosome amplification by p53 abrogation alone in these cells.

Metaphase checkpoint controls sense abnormalities of chromosome alignment during mitosis and prevent progression to anaphase until proper alignment has been attained. A number of proteins, including mad2, bub1, and bubR1, have been implicated in the metaphase checkpoint control in mammalian cells. Metaphase checkpoints have been shown, in various systems, to read loss of either spindle tension or microtubule attachment at the kinetochore. Characteristically, HeLa cells arrest in metaphase in response to low levels of microtubule inhibitors that leave an intact spindle and a metaphase plate. Here we show that the arrest induced by nanomolar vinblastine correlates with loss of tension at the kinetochore, and that in response the checkpoint proteins bub1 and bubR1 are recruited to the kinetochore but mad2 is not. mad2 remains competent to respond and is recruited at higher drug doses that disrupt spindle association with the kinetochores. Further, although mad2 forms a complex with cdc20, it does not associate with bub1 or bubR1. We conclude that mammalian bub1/bubR1 and mad2 operate as elements of distinct pathways sensing tension and attachment, respectively.