Caffeine promotes apoptosis in mitotic spindle checkpoint-arrested cells

The spindle assembly checkpoint arrests cells in mitosis when defects in mitotic spindle assembly or partitioning of the replicated genome are detected. This checkpoint blocks exit from mitosis until the defect is rectified or the cell initiates apoptosis. In this study we have used caffeine to identify components of the mechanism that signals apoptosis in mitotic checkpoint-arrested cells. Addition of caffeine to spindle checkpoint-arrested cells induced > 40% apoptosis within 5 h. It also caused proteasome-mediated destruction of cyclin B1, a corresponding reduction in cyclin B1/cdk1 activity, and reduction in NIPM-2 reactivity. However, cells retained MAD2 staining at the kinetochores, an indication of continued spindle checkpoint function. Blocking proteasome activity did not block apoptosis, but continued spindle checkpoint function was essential for apoptosis. After systematically eliminating all known targets, we have identified p21-activated kinase PAK1, which has an anti-apoptotic function in spindle checkpoint-arrested cells, as a target for caffeine inhibition. Knockdown of PAKI. also increased apoptosis in spindle checkpoint-arrested cells. This study demonstrates that the spindle checkpoint not only regulates mitotic exit but apoptosis in mitosis through the activity of PAKI.