Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines

Checkpoint mechanism plays a crucial role in ensuring genomic integrity dur ing cell cycle. Loss of checkpoint function is known to induce genomic inst ability and to alter ploidy of dividing cells. In this study, we examined m echanisms of hyperploid formation in glioma cells by treatment with nocodaz ole, which activates spindle assembly checkpoint by inhibiting microtubule polymerization. By prolonged nocodazole treatment, U251MG human glioma cell , which has a p53 mutation, underwent transient arrest at mitosis, and subs equently exited from mitotic arrest (termed 'mitotic slippage') followed by DNA replication without cytokinesis, resulting in hyperploid formation. Ad ditionally, the heterogeneity in the number of centrosomes per cell increas ed during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a tempera ture-sensitive p53 mutation, Ne found that the activation of p53 prevents h yperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases i ncluding cdc2, was found to enhance hyperploid formation in U251MG cells by accelerating the induction of mitotic slippage. Interestingly, inhibitors specific for cdc2 kinase prevented the G2 to M transition but did not accel erate mitotic slippage, suggesting that staurosporine-sensitive kinases oth er than cdc2 are required for maintenance of spindle assembly checkpoint. M oreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogati on of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.