The G₂ Checkpoint Is Maintained by Redundant Pathways

Abstract

While p53 activity is critical for a DNA damage-induced G₁ checkpoint, its role in the G₂ checkpoint has not been compelling because cells lacking p53 retain the ability to arrest in G₂ following DNA damage. Comparison between normal human foreskin fibroblasts (HFFs) and HFFs in which p53 was eliminated by transduction with human papillomavirus type 16 E6 showed that treatment with adriamycin initiated arrest in G₂ with active cyclin B/CDC2 kinase, regardless of p53 status. Both E6-transduced HFFs and control (LXSN)-transduced cells maintained a prolonged arrest in G₂; however cells with functional p53 extinguished cyclin B-associated kinase activity. Down regulation was mediated by p53-dependent transcriptional repression of the CDC2 and cyclin B promoters. In contrast, cells lacking p53 showed a prolonged G₂ arrest despite high levels of cyclin B/CDC2 kinase activity, at least some of which translocated into the nucleus. Furthermore, the G₂ checkpoint became attenuated as p53-deficient cells aged in culture. Thus, at late passage, E6-transduced HFFs entered mitosis following DNA damage, whereas the age-matched parental HFFs sustained a G₂ arrest. These results indicate that normal cells have p53-independent pathways to maintain DNA damage-induced G₂ arrest, which may be augmented by p53-dependent functions, and that cells lacking p53 are at greater risk of losing the pathway that protects against aneuploidy.

ACKNOWLEDGMENTS

T.M.P. was supported by training grant CA09515, K12 CA76930, and CA79629-01. J.A.B. and L.G. were supported by the Molecular and Cellular Biology Graduate Program. J.A.B. was also supported by an N.S.F. predoctoral fellowship. The work was funded by grant CA64975 from NCI to D.A.G.

We thank Bill Kaufmann and members of the Galloway laboratory for stimulating discussions, and we thank the Flow Cytometry and Image Analysis lab for help with FACS analysis.