Cancer Res Treat.  2006 Dec;38(4):224-228.

p53 Prevents Immature Escaping from Cell Cycle G2 Checkpoint Arrest through Inhibiting cdk2-dependent NF-Y Phosphorylation

• Yun UJ
• Park HD
• Shin DY

Abstract

PURPOSE
Recent studies have suggested that p53 regulates the G2 checkpoint in the cell cycle and this function is required for the maintenance of genomic integrity. In this study, we addressed a role of p53 in escaping from cell cycle G2 arrest following DNA damage.
MATERIALS AND METHODS
Cell cycle checkpoint arrest in the human colon cancer cell line HCT116 and its derivatives carry p53 or p21 deletions, were examined by FACS analysis, immunoprecipitation, Western blot and IP-kinase assay.
RESULTS
While the cells with functional p53 were arrested at both the G1 and G2 checkpoints, the p53-deficient cells failed to arrest at G1, but they were arrested at G2. However, the p53-deficient cells failed to sustain G2 checkpoint arrest and they entered mitosis earlier than did the p53-positive cells and so this resulted in extensive cell death. Cdc2 kinase becomes reactivated in p53-deficient cells in association with entry into mitosis, but not in the p53-positive cells. Upon DNA damage, the p21-deficient cells, like the p53-negative cells, not only failed to repress cdk2- dependent NF-Y phosphorylation, but they also failed to repress the expression of such cell cycle G2-regulatory genes as cdc2, cyclin B, RNR-R2 and cdc25C, which have all been previously reported as targets of NF-Y transcription factor.
CONCLUSION
p53 is essential to prevent immature escaping from cell cycle G2 checkpoint arrest through p21-mediated cdk2 inactivation, and this leads to inhibition of cdk2-dependent NF-Y phosphorylation and NF-Y dependent transcription of the cell cycle G2-rgulatory genes, including cdc2 and cyclin B.

Keyword

Tumor suppressor protein p53; Cell cycle; G2 phase; Cdc2 protein kinase; NF-Y protein

MeSH Terms

Blotting, Western
CCAAT-Binding Factor
CDC2 Protein Kinase
Cell Cycle Checkpoints
Cell Cycle*
Cell Death
Cell Line
Colonic Neoplasms
Cyclin B
DNA Damage
G2 Phase
Humans
Immunoprecipitation
Mitosis
Phosphorylation*
Phosphotransferases
Transcription Factors
Tumor Suppressor Protein p53
United Nations*
CCAAT-Binding Factor
CDC2 Protein Kinase
Cyclin B
Phosphotransferases
Transcription Factors
Tumor Suppressor Protein p53

Figure

• 

  Fig. 1 Cell cycle analysis of the p53-deficient cells and the p53-positive cells after DNA damage. HCT116 cells and its p53-knockout derivative (HCT116 p53-/-) were irradiated with γ-rays and then cultured for the indicated times. For flow cytometry analysis, those cells were trypsinized, fixed and stained with propidium iodide.

• 

  Fig. 2 p53 is essential to keep cdc2 kinase inactive after DNA damage. (A) Cell lysates that were prepared as in Fig. 1 were subjected to Western blot analysis with antibodies against the indicated proteins. (B) Cdc2 kinase assay was performed by immunoprecipitation with anti-cdc2 antibody and with using Histone H1 as a substrate.

• 

  Fig. 3 p21 is essential for repression of the cell cycle G2 regulatory genes after DNA damage. HCT116 cells and its p21-knockout derivates (p21-/-) were treated with doxorubicin for 24 h. The cellular lysates were subjected to Western blot analysis with antibodies against cyclin A, cyclin B, cdc2, RNR R2 and cdc25c.

• 

  Fig. 4 p21 is essential for dephosphorylation of NF-Y and cdk2 inactivation after DNA damage. HCT116 cells and its p21-knockout derivates (p21-/-) were transfected with Flag-YA and then treated with doxorubicin for 24 h. The cells expressing Flag-YA were labeled with [32P] orthophosphote and they were next visualized by autoradigraphy as previously described (13). The activities of cdk2 were measured by IP-kinase assay.

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