Mechanism of hyperploid cell formation induced by microtubule inhibiting drug in glioma cell lines.
Checkpoint mechanism plays a crucial role in ensuring genomic integrity during cell cycle. Loss of checkpoint function is known to induce genomic instability and to alter ploidy of dividing cells. In this study, we examined mechanisms of hyperploid formation in glioma cells by treatment with nocodazole, 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 subsequently exited from mitotic arrest (termed 'mitotic slippage') followed by DNA replication without cytokinesis, resulting in hyperploid formation. Additionally, the heterogeneity in the number of centrosomes per cell increased during the hyperploid formation, suggesting that these hyperploid cells have genomic instability. By employing LN382 glioma cell that has a temperature-sensitive p53 mutation, we found that the activation of p53 prevents hyperploid formation after the prolonged nocodazole treatment. Furthermore, staurosporine, an inhibitor for a broad range of serine/threonine kinases including 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 accelerate mitotic slippage, suggesting that staurosporine-sensitive kinases other than cdc2 are required for maintenance of spindle assembly checkpoint. Moreover, the enhancement of hyperploid formation by staurosporine was also blocked by p53-dependent G1 checkpoint. These results suggest that abrogation of G1 checkpoint is a critical factor for formation of hyperploid cells after the mitotic slippage.
Tsuiki
H
H
Department of Tumor Genetics and Biology, Kumamoto University School of Medicine, 2-2-1 Honjo, Kumamoto 860-0811, Japan.
Nitta
M
M
Tada
M
M
Inagaki
M
M
Ushio
Y
Y
Saya
H
H
D016203:CDC2 Protein Kinase / Q000037:antagonists & inhibitors; D005910:Glioma / Q000473:pathology*; D006801:Humans; D008870:Microtubules / Q000187:drug effects*; D008938:Mitosis / Q000187:drug effects*; D008940:Mitotic Index; D015739:Nocodazole / Q000494:pharmacology*; D011123:Polyploidy; D019311:Staurosporine / Q000494:pharmacology; D014407:Tumor Cells, Cultured; D016159:Tumor Suppressor Protein p53 / Q000235:genetics
D016159:Tumor Suppressor Protein p53; D016203:CDC2 Protein Kinase; D019311:Staurosporine; D015739:Nocodazole