Partial inhibition of Cdk1 in G₂ phase overrides the SAC and decouples mitotic events

Abstract

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin-dependent kinase 1 (Cdk1). In this study we utilized low doses of the Cdk1-specific inhibitor, RO3306 from early G₂ phase onwards. Addition of low doses of RO3306 in G₂ phase induced minor chromosome congression and segregation defects. In contrast, mild doses of RO3306 during G₂ phase resulted in cells entering an aberrant mitosis, with cells fragmenting centrosomes and failing to fully disassemble the nuclear envelope. Cells often underwent cytokinesis and metaphase simultaneously, despite the presence of an active spindle assembly checkpoint, which prevented degradation of cyclin B1 and securin, resulting in the random partitioning of whole chromosomes. This highly aberrant mitosis produced a significant increase in the proportion of viable polyploid cells present up to 3 days post-treatment. Furthermore, cells treated with medium doses of RO3306 were only able to reach the threshold of Cdk1 substrate phosphorylation required to initiate nuclear envelope breakdown, but failed to reach the levels of phosphorylation required to correctly complete pro-metaphase. Treatment with low doses of Okadaic acid, which primarily inhibits PP2A, rescued the mitotic defects and increased the number of cells that completed a normal mitosis. This supports the current model that PP2A is the primary phosphatase that counterbalances the activity of Cdk1 during mitosis. Taken together these results show that continuous and subtle disruption of Cdk1 activity from G₂ phase onwards has deleterious consequences on mitotic progression by disrupting the balance between Cdk1 and PP2A.

Keywords: :

• Cdk1
• mitosis
• SAC
• PP2A
• cyclin B1
• RO3306

Disclosure of Potential Conflicts of Interest

No potential conflicts of interest were disclosed.

Acknowledgments

We thank Sylvain De Rossi (Montpellier RIO Imaging), Dr Will Hughes (Pieter Huveneers Molecular Imaging Unit, Garvan Institute), Dr Kim Moran-Jones, and Dr Paul Timpson for helpful comments. This work was supported by the Cancer Institute of NSW, and the Ligue Nationale Contre le Cancer (Equipe Labelisée) and Agence Nationale de la Recherche (ANR) 2010 BLAN 120701. A.B. is a CINSW FRL fellow and C.E.C. is an NBCF Australia fellow and CI NSW CD fellow.