Human topoisomerase II function, tyrosine phosphorylation and cell cycle checkpoints

Area of Science:

• Cell Biology
• Molecular Biology
• Genetics

Background:

• Diploid human fibroblasts possess three DNA damage-responsive cell cycle checkpoints: G1 arrest, S phase replicon initiation inhibition, and G2/M delay.
• Progression from G2 to mitosis involves cyclin B/Cdk1 kinase activity, regulated by inhibitory Tyr15 phosphorylation and activating phosphatase Cdc25C.
• DNA damage induces a G2 delay by inhibiting the Cdc25C-mediated activation loop of cyclin B/Cdk1, preventing mitotic entry.

Purpose of the Study:

• To investigate the mechanisms of DNA damage-responsive cell cycle checkpoints in human fibroblasts.
• To explore the role of the isoflavone genistein and topoisomerase poisons in modulating these checkpoints.
• To understand how these agents may lead to genetic instability.

Main Methods:

• Observation of cell cycle progression in human fibroblasts following DNA damage induction.
• Treatment with genistein to inhibit tyrosine kinases involved in Cdk1 phosphorylation.
• Treatment with amsacrine, a type II DNA topoisomerase poison, to assess S phase checkpoint response.

Main Results:

• DNA damage delays mitosis by inhibiting cyclin B/Cdk1 activation via suppression of the Cdc25C feedback loop.
• Genistein inhibits a tyrosine kinase (p56/p53lyn) that phosphorylates Cdk1, and also inhibits type II DNA topoisomerase, triggering checkpoints.
• Amsacrine treatment rapidly inhibits replicon initiation, with the checkpoint response decaying over time; topoisomerase II poisons cause chromosomal aberrations.

Conclusions:

• The G2/M delay is mediated by the inhibition of cyclin B/Cdk1 activation following DNA damage.
• Genistein and other topoisomerase poisons can disrupt cell cycle control by inducing DNA damage and potentially inactivating the ATM gene.
• Disruption of cell cycle checkpoints by these agents can lead to genetic instability and potentially cancer.