title: ATM and the Mre11-Rad50-Nbs1 complex respond to nucleoside analogue-induced stalled replication forks and contribute to drug resistance.
abstract: The Mre11-Rad50-Nbs1 complex and autophosphorylated Ser(1981)-ATM are involved in recognizing and repairing DNA damage, such as double-strand breaks (DSB). However, the role of these factors in response to stalled replication forks is not clear. Nucleoside analogues are agents that are incorporated into DNA during replication, which cause stalling of replication forks. The molecular mechanisms that sense these events may signal for DNA repair and contribute to survival but are poorly understood. Cellular responses to both DSBs and stalled replication forks are marked by H2AX phosphorylation on Ser(139) (gamma-H2AX), which forms nuclear foci at sites of DNA damage. Here, concentrations of the nucleoside analogues 1-beta-d-arabinofuranosylcytosine (cytarabine; ara-C), gemcitabine, and troxacitabine, which inhibited DNA synthesis by 90% within 2 hours, were determined for each agent. Using gamma-H2AX as a marker for changes in chromatin structure, we show that Mre11, Rad50, Nbs1, and phosphorylated ATM respond to nucleoside analogue-induced stalled replication forks by forming nuclear foci that colocalize with gamma-H2AX within 2 hours. Because neither DSBs nor single-strand breaks were detectable after nucleoside analogue exposure, we conclude that this molecular response is not due to the presence of DNA breaks. Deficiencies in ATM, Mre11, or Rad50 led to a 2- to 5-fold increase in clonogenic sensitization to gemcitabine, whereas Nbs1 and H2AX deficiency did not affect reproductive growth. Taken together, these results suggest that ATM, Mre11, and Rad50 are required for survival after replication fork stalling, whereas Nbs1 and H2AX are inconsequential.
authors.affiliation: Department of Experimental Therapeutics, The University of Texas MD Anderson Cancer Center, Houston Graduate School of Biomedical Sciences, Houston, Texas 77030, USA.
authors.forename: Brett
authors.initials: B
authors.lastname: Ewald
authors.forename: Deepa
authors.initials: D
authors.lastname: Sampath
authors.forename: William
authors.initials: W
authors.lastname: Plunkett
mesh_terms: D017766:Acid Anhydride Hydrolases; D000818:Animals; D000970:Antineoplastic Agents / Q000627:therapeutic use; D064007:Ataxia Telangiectasia Mutated Proteins; D018797:Cell Cycle Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D002470:Cell Survival / Q000187:drug effects / Q000235:genetics; D002478:Cells, Cultured; D045643:DNA Repair Enzymes / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D004261:DNA Replication / Q000187:drug effects*; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D003841:Deoxycytidine / Q000031:analogs & derivatives; D019008:Drug Resistance, Neoplasm / Q000235:genetics*; D006801:Humans; D007951:Leukemia, Myeloid / Q000188:drug therapy / Q000235:genetics* / Q000378:metabolism / Q000473:pathology; D000076228:MRE11 Homologue Protein; D051379:Mice; D008954:Models, Biological; D046912:Multiprotein Complexes / Q000235:genetics / Q000378:metabolism / Q000502:physiology; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism / Q000502:physiology*; D009705:Nucleosides / Q000737:chemistry / Q000494:pharmacology*; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000502:physiology*; D014410:Tumor Stem Cell Assay; D025521:Tumor Suppressor Proteins / Q000235:genetics / Q000502:physiology*
chemical_list: D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; C121766:MRE11 protein, human; D046912:Multiprotein Complexes; C112156:NBN protein, human; D009687:Nuclear Proteins; D009705:Nucleosides; D025521:Tumor Suppressor Proteins; D003841:Deoxycytidine; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; D017346:Protein Serine-Threonine Kinases; D000076228:MRE11 Homologue Protein; D017766:Acid Anhydride Hydrolases; C112158:RAD50 protein, human; D045643:DNA Repair Enzymes