Abstract
The dual specificity phosphatase Cdc25B is capable of inhibiting cellular proliferation, and this occurs in a manner dependent upon its catalytic activity. Here it is shown that this is accompanied by inappropriate cyclin-dependent kinase activation and premature mitotic entry, leading to both p53-dependent and independent checkpoints. Forced expression of Cdc25B inappropriately up-regulated the activity of Cdk1 and Cdk2, by reducing levels of inhibitory phosphorylation. In cells lacking p14ARF, p53 is induced, and components of the ATM and ATR pathways are activated. Cdc25B triggers cell cycle arrest in the G(1) and G(2) phases that is p53- and p21-dependent and is inhibited by caffeine. Cdc25B also causes cells with an S phase DNA content to enter mitosis prematurely in a p53-independent manner. Synchronization of cells with aphidicolin results in these cells undergoing apoptosis. Thus, inappropriate cell cycle progression and premature mitotic entry via dysregulation of cyclin-dependent kinases results in activation of both p53-dependent and independent responses. Because Cdc25B is known to have oncogenic activity, this provides insight into the multistep nature of cancer development and why there is p53 loss during tumorigenesis.
Highlights
APRIL 3, 2009 VOLUME 284 NUMBER 14 tyrosine 15
It was shown that Cdc25B, as has been shown for other oncogenes, is capable of inhibiting cellular proliferation, and this occurs in a manner dependent upon its catalytic activity [10]
A p53-null tumor cell line, EJ, shows a similar cell cycle profile after infection with Ad-25B. These results indicate that the cell cycle arrest in the G1 and G2 phases caused by overexpression of Cdc25B are, p53-dependent
Summary
Cell Culture—Human U2OS, 293T, and Saos cells were grown in monolayer in Dulbecco’s modified Eagle’s medium (Invitrogen) supplemented with 10% heat-inactivated fetal calf serum (Invitrogen) and 10% penicillin/streptomycin (Invitrogen) at 5% CO2 and 37 °C. After washing in PBS, the cells were blocked in 3% bovine serum albumin for 10 min prior to incubation with a buffer containing 3% bovine serum albumin/PBS and 1:1000 dilution of mouse anti-␥H2AX (Upstate, Cell Signaling Solutions) for 1 h. The cells were washed in PBS and subsequently incubated with a 1:2000 dilution of Alexa Fluor 568 goat anti-mouse IgG (Molecular Probes, Invitrogen Detection Technologies) in 3% bovine serum albumin/PBS for 1 h. When cells were double-stained, Alexa Fluor 647 goat anti-rabbit IgG (Molecular Probes, Invitrogen Detection Technologies) was included at a dilution of 1:2000. These cells exited S phase prematurely and inappropriately entered
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