Abstract
An ATR-dependent G(2) checkpoint responds to inhibition of topoisomerase II and delays entry into mitosis by sustaining nuclear exclusion of cyclin B1-Cdk1 complexes. Here we report that induction of this checkpoint with ICRF-193, a topoisomerase II catalytic inhibitor that does not cause DNA damage, was associated with an ATR-dependent inhibition of polo-like kinase 1 (Plk1) kinase activity and a decrease in cyclin B1 phosphorylation. Expression of constitutively active Plk1 but not wild type Plk1 reversed ICRF-193-induced mitotic delay in HeLa cells, suggesting that Plk1 kinase activity is important for the checkpoint response to ICRF-193. G(2)/M synchronized normal human fibroblasts, when treated with ICRF-193, showed a decrease in cyclin B1 phosphorylation and Plk1 kinase activity despite high cyclin B1-Cdk1 kinase activity. G(2) fibroblasts that were treated with caffeine to override the checkpoint response to ICRF-193 displayed a high incidence of chromosomal aberrations. Taken together, these results suggest that ATR-dependent inhibition of Plk1 kinase activity may be one mechanism to regulate cyclin B1 phosphorylation and sustain nuclear exclusion during the G(2) checkpoint response to topoisomerase II inhibition. Moreover, the results demonstrate an important role for the topoisomerase II-dependent G(2) checkpoint in the preservation of human genomic stability.
Highlights
Cell cycle checkpoints are surveillance systems that ensure orderly and timely replication and segregation of the genome
Because polo-like kinase 1 (Plk1) activity is regulated by the DNA damage checkpoint and one function of Plk1 involves regulation of cyclin B1 localization, we determined whether ICRF-193-induced mitotic delay was associated with an inhibition of Plk1 activity
The findings presented here demonstrate that Plk1 kinase activity and cyclin B1 phosphorylation are regulated by a topoisomerase II-dependent G2 checkpoint
Summary
Polo-like kinase 1; FBS, fetal bovine serum; PBS, phosphate-buffered saline; GFP, green fluorescent protein. The topoisomerase II-dependent G2 checkpoint response to ICRF-193 was recently reported to be distinct from the DNA damage G2 checkpoint as it was ATM-independent and did not appear to be enforced through inhibition of cyclin B1-Cdk kinase activity. G2 cells arrested with ICRF193 displayed reduced serine phosphorylation of cyclin B1 in vivo and reduced kinase activity on cyclin B1 in cell-free extracts. Taken together these data support a model for the topoisomerase II-dependent G2 checkpoint whereby ATR signaling inhibits Plk activity, which in turn blocks the phosphorylation of cyclin B1 and prevents the accumulation of nuclear cyclin B1-Cdk complexes
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