Inhibition of human Chk1 causes increased initiation of DNA replication, phosphorylation of ATR targets, and DNA breakage.
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.
Syljuåsen
Randi G
RG
Institute of Cancer Biology, Department of Cell Cycle and Cancer, Danish Cancer Society, Strandboulevarden 49, 2100 Copenhagen, Denmark.
Sørensen
Claus Storgaard
CS
Hansen
Lasse Tengbjerg
LT
Fugger
Kasper
K
Lundin
Cecilia
C
Johansson
Fredrik
F
Helleday
Thomas
T
Sehested
Maxwell
M
Lukas
Jiri
J
Bartek
Jiri
J
D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D002868:Chromosomal Proteins, Non-Histone / Q000378:metabolism; D004249:DNA Damage / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects / Q000502:physiology*; D004277:DNA, Single-Stranded / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000378:metabolism; D006657:Histones / Q000378:metabolism; D006801:Humans; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000235:genetics / Q000494:pharmacology* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000187:drug effects / Q000502:physiology*; D011687:Purines / Q000494:pharmacology; D034741:RNA, Small Interfering / Q000235:genetics / Q000494:pharmacology; D051716:Replication Protein A; D000077546:Roscovitine; D019311:Staurosporine / Q000031:analogs & derivatives* / Q000494:pharmacology; D016159:Tumor Suppressor Protein p53 / Q000378:metabolism; D000098550:Structural Maintenance of Chromosome Protein 1
C113571:CDC45 protein, human; D018797:Cell Cycle Proteins; D002868:Chromosomal Proteins, Non-Histone; D004277:DNA, Single-Stranded; D004268:DNA-Binding Proteins; C468783:H2AX protein, human; D006657:Histones; D047428:Protein Kinase Inhibitors; D011687:Purines; D034741:RNA, Small Interfering; C497167:RPA1 protein, human; D051716:Replication Protein A; D016159:Tumor Suppressor Protein p53; D000098550:Structural Maintenance of Chromosome Protein 1; D000077546:Roscovitine; D002110:Caffeine; C054852:7-hydroxystaurosporine; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases; D019311:Staurosporine