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Molecular and Cellular Biology, May 2005, p. 3553-3562, Vol. 25, No. 9
0270-7306/05/$08.00+0     doi:10.1128/MCB.25.9.3553-3562.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

Inhibition of Human Chk1 Causes Increased Initiation of DNA Replication, Phosphorylation of ATR Targets, and DNA Breakage

Randi G. Syljuåsen,1,{dagger} Claus Storgaard Sørensen,1,{dagger},{ddagger} Lasse Tengbjerg Hansen,2 Kasper Fugger,1 Cecilia Lundin,3 Fredrik Johansson,3 Thomas Helleday,3,4 Maxwell Sehested,5 Jiri Lukas,1 and Jiri Bartek1*

Institute of Cancer Biology, Danish Cancer Society,1 Institute of Molecular Pathology, University of Copenhagen,2 Department of Pathology, Rigshospitalet 5444, Copenhagen, Denmark,5 Department of Genetics Microbiology and Toxicology, Stockholm University, Stockholm, Sweden,3 The Institute for Cancer Studies, University of Sheffield, Sheffield, United Kingdom4

Received 12 July 2004/ Returned for modification 26 August 2004/ Accepted 4 January 2005

Human checkpoint kinase 1 (Chk1) is an essential kinase required to preserve genome stability. Here, we show that Chk1 inhibition by two distinct drugs, UCN-01 and CEP-3891, or by Chk1 small interfering RNA (siRNA) leads to phosphorylation of ATR targets. Chk1-inhibition triggered rapid, pan-nuclear phosphorylation of histone H2AX, p53, Smc1, replication protein A, and Chk1 itself in human S-phase cells. These phosphorylations were inhibited by ATR siRNA and caffeine, but they occurred independently of ATM. Chk1 inhibition also caused an increased initiation of DNA replication, which was accompanied by increased amounts of nonextractable RPA protein, formation of single-stranded DNA, and induction of DNA strand breaks. Moreover, these responses were prevented by siRNA-mediated downregulation of Cdk2 or the replication initiation protein Cdc45, or by addition of the CDK inhibitor roscovitine. We propose that Chk1 is required during normal S phase to avoid aberrantly increased initiation of DNA replication, thereby protecting against DNA breakage. These results may help explain why Chk1 is an essential kinase and should be taken into account when drugs to inhibit this kinase are considered for use in cancer treatment.

* Corresponding author. Mailing address: Institute of Cancer Biology, Department of Cell Cycle and Cancer, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen, Denmark. Phone: (45) 35257357. Fax: (45) 35257721. E-mail: bartek{at}biobase.dk.

{dagger} R.G.S. and C.S.S. contributed equally to this study.

{ddagger} Present address: Biotech Research and Innovation Centre, DK-2100 Copenhagen, Denmark.

Molecular and Cellular Biology, May 2005, p. 3553-3562, Vol. 25, No. 9
0022-538X/05/$08.00+0     doi:10.1128/MCB.25.9.3553-3562.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



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