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

Samuel McNeely1, Chiara Conti, Tahir Sheikh

  • 1Department of Medicine, Memorial Sloan-Kettering Cancer Center, New York, NY, USA.

Cell Cycle (Georgetown, Tex.)
|February 18, 2010
PubMed

Insights

Inhibiting Checkpoint kinase 1 (Chk1) with AZD7762 enhances gemcitabine chemotherapy by destabilizing replication forks and causing DNA breaks. This combination therapy may be effective against tumors with DNA repair defects.

Area of Science:

  • Molecular Biology
  • Cancer Research
  • Cell Biology

Background:

  • Checkpoint kinase 1 (Chk1) is crucial for cell cycle checkpoints and DNA repair following genotoxic stress.
  • Inhibiting Chk1 is a promising strategy to enhance chemotherapy efficacy.
  • AZD7762 is an ATP-competitive inhibitor targeting Chk1/2 kinases.

Purpose of the Study:

  • To investigate the mechanisms by which AZD7762 potentiates gemcitabine cytotoxicity.
  • To identify the specific DNA damage response pathways involved in AZD7762 and gemcitabine combination therapy.
  • To explore the potential of this combination therapy in specific cancer types.

Main Methods:

  • Treatment of cells with AZD7762 and gemcitabine.
  • Analysis of replication fork dynamics and stability using DNA fiber combing.
  • Measurement of DNA breaks via comet assay.
  • Assessment of kinase involvement (ATM, DNA-PK, ATR) in gammaH2AX induction.
  • Evaluation of sensitivity in cells deficient in DNA double-strand break (DSB) repair.
  • Monitoring cell cycle progression and mitotic entry.
  • Investigation of apoptosis induction and prevention.

Main Results:

  • AZD7762 induces gammaH2AX in gemcitabine-treated cells by altering replication fork dynamics and stability.
  • ATM and DNA-PK, not ATR, mediate gammaH2AX induction, suggesting conversion of stalled forks to DSBs.
  • Cells deficient in DSB repair (BRCA2, XRCC3, DNA-PK) show increased sensitivity to the combination therapy.
  • AZD7762 causes premature mitosis in gemcitabine-treated cells, leading to apoptosis.
  • Combination therapy involves origin firing activation, replication fork destabilization, and entry into lethal mitosis.

Conclusions:

  • Chemosensitization by Chk1 inhibition involves multiple cellular events: origin firing, replication fork collapse, and premature mitotic entry.
  • The combination of AZD7762 and gemcitabine may be particularly effective in tumors with defects in DNA repair pathways.
  • Targeting Chk1 offers a viable strategy to overcome gemcitabine resistance and enhance anti-cancer effects.

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