Endogenous γ-H2AX-ATM-Chk2 Checkpoint Activation in Bloom's Syndrome Helicase–Deficient Cells Is Related to DNA Replication Arrested Forks

  • V. Ashutosh Rao
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;
  • Chiara Conti
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;
  • Josee Guirouilh-Barbat
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;
  • Asako Nakamura
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;
  • Ze-Hong Miao
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;
  • Sally L. Davies
    3Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
  • Barbara Saccá
    2Genome Stability Laboratory, Pasteur Institute, Paris, France; and
  • Ian D. Hickson
    3Cancer Research UK Laboratories, Weatherall Institute of Molecular Medicine, University of Oxford, John Radcliffe Hospital, Oxford, United Kingdom
  • Aaron Bensimon
    2Genome Stability Laboratory, Pasteur Institute, Paris, France; and
  • Yves Pommier
    1Laboratory of Molecular Pharmacology, Center for Cancer Research, National Cancer Institute, U.S. Department of Health and Human Services, Bethesda, Maryland;

抄録

<jats:title>Abstract</jats:title> <jats:p>The Bloom syndrome helicase (BLM) is critical for genomic stability. A defect in BLM activity results in the cancer-predisposing Bloom syndrome (BS). Here, we report that BLM-deficient cell lines and primary fibroblasts display an endogenously activated DNA double-strand break checkpoint response with prominent levels of phosphorylated histone H2AX (γ-H2AX), Chk2 (pT68Chk2), and ATM (pS1981ATM) colocalizing in nuclear foci. Interestingly, the mitotic fraction of γ-H2AX foci did not seem to be higher in BLM-deficient cells, indicating that these lesions form transiently during interphase. Pulse labeling with iododeoxyuridine and immunofluorescence microscopy showed the colocalization of γ-H2AX, ATM, and Chk2 together with replication foci. Those foci costained for Rad51, indicating homologous recombination at these replication sites. We therefore analyzed replication in BS cells using a single molecule approach on combed DNA fibers. In addition to a higher frequency of replication fork barriers, BS cells displayed a reduced average fork velocity and global reduction of interorigin distances indicative of an elevated frequency of origin firing. Because BS is one of the most penetrant cancer-predisposing hereditary diseases, it is likely that the lack of BLM engages the cells in a situation similar to precancerous tissues with replication stress. To our knowledge, this is the first report of high ATM-Chk2 kinase activation and its linkage to replication defects in a BS model. (Mol Cancer Res 2007;5(7):713–24)</jats:p>

収録刊行物

  • Molecular Cancer Research

    Molecular Cancer Research 5 (7), 713-724, 2007-07-01

    American Association for Cancer Research (AACR)

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