Chk1 inhibition after replicative stress activates a double strand break response mediated by ATM and DNA-dependent protein kinase

Samuel McNeely, Chiara Conti, Tahir Sheikh, Himali Patel, Sonya Zabludoff, Yves Pommier, Gary Schwartz, Archie Tse

Research output: Contribution to journalArticlepeer-review

77 Scopus citations

Abstract

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 γH2AX 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 γH2AX 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 G1/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.

Original languageEnglish
Pages (from-to)995-1004
Number of pages10
JournalCell Cycle
Volume9
Issue number5
DOIs
StatePublished - 1 Mar 2010
Externally publishedYes

Keywords

  • ATM
  • AZD7762
  • Chk1
  • DNA-PK
  • Gemcitabine

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