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Wild-type TP53 inhibits G(2)-phase checkpoint abrogation and radiosensitization induced by PD0166285, a WEE1 kinase
1Department of Radiation Oncology, University of Michigan, Ann Arbor Michigan 48109, USA.
Abstract:
The WEE1 protein kinase carries out the inhibitory phosphorylation of CDC2 on tyrosine 15 (Tyr15), which is required for activation of the G(2)-phase checkpoint in response to DNA damage. PD0166285 is a newly identified WEE1 inhibitor and is a potential selective G(2)-phase checkpoint abrogator. To determine the role of TP53 in PD0166285-induced G(2)-phase checkpoint abrogation, human H1299 lung carcinoma cells expressing a temperature-sensitive TP53 were used. Upon exposure to gamma radiation, cells cultured under nonpermissive conditions (TP53 mutant conformation) underwent G(2)-phase arrest. However, under permissive conditions (TP53 wild-type conformation), PD0166285 greatly inhibited the accumulation of cells in G(2) phase. This abrogation was accompanied by a nearly complete blockage of Tyr15 phosphorylation of CDC2, an increased activity of CDC2 kinase, and an enhanced sensitivity to radiation. However, under permissive conditions (TP53 wild-type conformation), PD0166285 neither disrupted the G(2)-phase arrest nor increased cell death. The compound inhibited Tyr15 phosphorylation only partially and did not activate CDC2 kinase activity. To understand the potential mechanism(s) by which TP53 inhibits PD0166285-induced G(2)-phase checkpoint abrogation, two TP53 target proteins, 14-3-3rho and CDKN1A (also known as p21), that are known to be involved in G(2)-phase checkpoint control in other cell models were examined. It was found that 14-3-3rho was not expressed in H1299 cells, and that although CDKN1A did associate with CDC2 to form a complex, the level of CDKN1A associated with CDC2 was not increased in response to radiation or to PD0166285. The level of cyclin B1, required for CDC2 activity, was decreased in the presence of functional TP53. Thus inhibition of PD0166285-induced G(2)-phase checkpoint abrogation by TP53 was achieved at least in part through partial blockage of CDC2 dephosphorylation of Tyr15 and inhibition of cyclin B1 expression.
Insights
TP53 suppresses the WEE1 inhibitor PD0166285’s ability to abrogate the G2-phase checkpoint. Functional TP53 partially blocks CDC2 dephosphorylation and reduces cyclin B1, hindering checkpoint abrogation and enhancing radiation sensitivity.
Area of Science:
- Cell cycle regulation
- DNA damage response
- Cancer therapeutics
Background:
- WEE1 kinase phosphorylates CDC2 on Tyr15, crucial for the G2-phase DNA damage checkpoint.
- PD0166285 is a WEE1 inhibitor investigated as a G2-phase checkpoint abrogator.
- The role of TP53 in PD0166285-mediated checkpoint abrogation requires elucidation.
Purpose of the Study:
- To determine the role of TP53 in PD0166285-induced G2-phase checkpoint abrogation.
- To investigate the molecular mechanisms by which TP53 influences PD0166285 efficacy.
Main Methods:
- Utilized human H1299 lung carcinoma cells with temperature-sensitive TP53.
- Exposed cells to gamma radiation and treated with PD0166285 under permissive (wild-type TP53) and nonpermissive (mutant TP53) conditions.
- Assessed G2-phase arrest, CDC2 Tyr15 phosphorylation, CDC2 kinase activity, and levels of TP53 target proteins (14-3-3rho, CDKN1A, cyclin B1).
Main Results:
- In cells with mutant TP53, PD0166285 abrogated the G2-arrest, blocked CDC2 Tyr15 phosphorylation, increased CDC2 kinase activity, and enhanced radiation sensitivity.
- In cells with wild-type TP53, PD0166285 failed to disrupt G2-arrest or increase cell death, with only partial inhibition of Tyr15 phosphorylation and no CDC2 kinase activation.
- TP53 functional status influenced cyclin B1 levels, which were decreased in the presence of wild-type TP53.
Conclusions:
- TP53 inhibits PD0166285-induced G2-phase checkpoint abrogation.
- This inhibition is mediated, at least partly, by partial blockage of CDC2 dephosphorylation at Tyr15 and reduced cyclin B1 expression.
- Understanding TP53's role is critical for optimizing WEE1 inhibitor-based cancer therapies.
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