Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase.
Checkpoint kinase 1 (Chk1) regulates cell cycle checkpoints and DNA damage repair in response to genotoxic stress. Inhibition of Chk1 is an emerging strategy for potentiating the cytotoxicity of chemotherapeutic drugs. Here, we demonstrate that AZD7762, an ATP -competitive Chk1/2 inhibitor induces gammaH2AX in gemcitabine-treated cells by altering both dynamics and stability of replication forks, allowing the firing of suppressed replication origins as measured by DNA fiber combing and causing a dramatic increase in DNA breaks as measured by comet assay. Furthermore, we identify ATM and DNA-PK, rather than ATR, as the kinases mediating gammaH2AX induction, suggesting AZD7762 converts stalled forks into double strand breaks (DSBs). Consistent with DSB formation upon fork collapse, cells deficient in DSB repair by lack of BRCA2, XRCC3 or DNA-PK were selectively more sensitive to combined AZD7762 and gemcitabine. Checkpoint abrogation by AZD7762 also caused premature mitosis in gemcitabine-treated cells arrested in G(1)/early S-phase. Prevention of premature mitotic entry via Cdk1 siRNA knockdown suppressed apoptosis. These results demonstrate that chemosensitization of gemcitabine by Chk1 inhibition results from at least three cellular events, namely, activation of origin firing, destabilization of stalled replication forks and entry of cells with damaged DNA into lethal mitosis. Additionally, the current study indicates that the combination of Chk1 inhibitor and gemcitabine may be particularly effective in targeting tumors with specific DNA repair defects.
McNeely
Samuel
S
Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.
Conti
Chiara
C
Sheikh
Tahir
T
Patel
Himali
H
Zabludoff
Sonya
S
Pommier
Yves
Y
Schwartz
Gary
G
Tse
Archie
A
D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000378:metabolism*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D020552:Comet Assay; D053903:DNA Breaks, Double-Stranded; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000378:metabolism*; D003841:Deoxycytidine / Q000031:analogs & derivatives / Q000494:pharmacology; D016193:G1 Phase; D006657:Histones / Q000378:metabolism; D006801:Humans; D008938:Mitosis; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000737:chemistry / Q000378:metabolism*; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering / Q000378:metabolism; D016196:S Phase; D013876:Thiophenes / Q000494:pharmacology; D025521:Tumor Suppressor Proteins / Q000378:metabolism*; D014508:Urea / Q000031:analogs & derivatives / Q000494:pharmacology; D000093542:Gemcitabine
C532363:3-(carbamoylamino)-5-(3-fluorophenyl)-N-(3-piperidyl)thiophene-2-carboxamide; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D006657:Histones; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D013876:Thiophenes; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; D014508:Urea; D011494:Protein Kinases; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D051747:DNA-Activated Protein Kinase; D017346:Protein Serine-Threonine Kinases; D000093542:Gemcitabine