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Severe levels of hypoxia (oxygen concentrations of less that 0.02%) have been shown to induce a rapid S-phase arrest. The mechanism behind hypoxia-induced S-phase arrest is unclear, we show here that it was not mediated by a shortage of nucleosides and was not dependent on p53, p21 or Hif 1alpha status. The drugs aphidicolin and hydroxyurea both induce rapid replication arrest and have been used throughout the literature to study the ATR-mediated response to stalled replication. We have shown previously that hypoxia induces ATR-dependent phosphorylation of p53, Chk1 and histone H2AX. Using comet-assays to detect DNA-damage we found that both aphidicolin and hydroxyurea induced significant levels of DNA-damage while hypoxia did not. Here we show that like aphidicolin and hydroxyurea, hypoxia induces phosphorylation of Nbs1 at serine 343 and Rad17 serine 645. Hypoxia-dependent phosphorylation of Nbs1 and Rad17 was ATM-independent and therefore likely to be a result of the ATR kinase activity. In contrast, p53 was phosphorylated differentially in response to the three treatments considered here. p53 was phosphorylated at serine 15 in response to all three treatments but was only phosphorylated at serine 20 in response to the drug treatments. We propose that treatment with either aphidicolin or hydroxyurea leads to not only replication arrest but also DNA-damage and therefore both ATM and ATR-mediated signaling. In contrast replication arrest induced by severe hypoxia is sensed exclusively through ATR, with ATM only having a role to play after re-oxygenation.

Type

Journal article

Journal

Mutat Res

Publication Date

27/11/2003

Volume

532

Pages

205 - 213

Keywords

Animals, Aphidicolin, Ataxia Telangiectasia Mutated Proteins, Cell Cycle Proteins, Cell Hypoxia, Cells, Cultured, Comet Assay, DNA Damage, DNA Replication, DNA-Binding Proteins, Enzyme Inhibitors, Fibroblasts, Humans, Hydroxyurea, Hypoxia-Inducible Factor 1, alpha Subunit, Mice, Mice, Knockout, Nuclear Proteins, Phosphorylation, Protein-Serine-Threonine Kinases, S Phase, Transcription Factors, Tumor Suppressor Protein p53, Tumor Suppressor Proteins