Chk1 Mediates S and G2 Arrests through Cdc25A Degradation in Response to DNA-damaging Agents

Zhan Xiao,Zehan Chen,Angelo H Gunasekera,Thomas J Sowin,Saul H Rosenberg,Steve Fesik,Haiying Zhang

doi:10.1074/jbc.m300229200

Abstract

UV and ionizing radiation (IR) activate DNA damage checkpoints and induce Cdc25A degradation (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429; Falck, J., Mailand, N., Syljuasen, R. G., Bartek, J., and Lukas J. (2001) Nature 410, 842-847). The degradation of Cdc25A is abrogated by caffeine, which implicates Chk1 as the potential mediator (Mailand, N., Falck, J., Lukas, C., Syljuasen, R. G., Welcker, M., Bartek, J., and Lukas, J. (2000) Science 288, 1425-1429). However, the involvement of Chk1 is far from clear, because caffeine is a rather nonspecific inhibitor of the ATR/Chk1 signaling pathway. Additionally, it is not known whether DNA-damaging drugs commonly used in chemotherapy, which may activate different signal transduction pathways than UV or IR, also confer Cdc25A degradation. Herein, we show that camptothecin and doxorubicin, two widely used topoisomerase inhibitors conferring S and G2 arrest, respectively, cause the degradation of Cdc25A. Using a small interfering RNA that enables the specific elimination of Chk1 expression, we show that the observed proteolysis of Cdc25A is mediated through Chk1. Moreover, Cdc25A overexpression abrogates the Chk1-mediated degradation and overcomes the doxorubicin-induced G2 arrest through dephosphorylation and activation of Cdc2/Cdk1 in a dose-dependent manner. These results suggest that: (a) Cdc25A is involved in the G2/M transition in addition to its commonly accepted effect on G1/S progression, and (b) Chk1 mediates both S and G2 checkpoint and is thus a more ubiquitous cell cycle checkpoint mediator than previously thought.

Highlights

DNA damage caused by different agents induces cell cycle arrest at G1, S, or G2, thereby preventing replication of damaged DNA or aberrant mitosis until the damage is adequately repaired
To investigate if this finding can be extended to DNA damageinduced arrest at other phases of the cell cycle, we studied the effect of camptothecin, a clinically relevant topoisomerase I inhibitor conferring S phase arrest
We confirmed that cells treated with camptothecin were mostly arrested in S phase (Fig. 3B and data not shown), further suggesting that Chk1 may mediate S checkpoint by targeting Cdc25A for proteolysis

Summary

Introduction

DNA damage caused by different agents induces cell cycle arrest at G1, S, or G2, thereby preventing replication of damaged DNA or aberrant mitosis until the damage is adequately repaired. These regulatory mechanisms are known as cell cycle checkpoints and involve an intricate network of protein kinase signaling pathways. By abrogating the G2 checkpoint, normal cells can still arrest in the G1 phase and repair the DNA damage; whereas, tumor cells that lack the G1 checkpoint will undergo mitotic catastrophe and eventually undergo apoptosis. Chk Mediates Both S and G2 Checkpoints role in the DNA damage-induced G2 checkpoint, Chk has become an attractive target for this approach, and Chk inhibitors are being developed to potentiate the effects of genotoxic chemotherapeutics on p53-null tumors

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Conclusion