Partial inhibition of Cdk1 in G₂phase overrides the SAC and decouples mitotic events

TLDR

Entry and progression through mitosis has traditionally been linked directly to the activity of cyclin‑dependent kinase 1 (Cdk1). The study aimed to investigate the effects of low doses of the Cdk1 inhibitor RO3306 applied from early G2 phase onwards. The authors applied low doses of RO3306 to cells starting in early G2 phase. Low‑dose RO3306 treatment from early G2 induced chromosome congression/segregation defects, aberrant mitosis with fragmented centrosomes, failed nuclear envelope breakdown, simultaneous cytokinesis and metaphase, random chromosome partitioning, and a rise in viable polyploid cells, while medium doses only allowed nuclear envelope breakdown but not full pro‑metaphase; rescue with Okadaic acid confirmed PP2A as the key phosphatase opposing Cdk1, demonstrating that subtle Cdk1 inhibition from G2 disturbs the Cdk1/PP2A balance and mitotic progression.

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.