title: DNA protein kinase-dependent G2 checkpoint revealed following knockdown of ataxia-telangiectasia mutated in human mammary epithelial cells.
abstract: Members of the phosphatidylinositol 3-kinase-related kinase family, in particular the ataxia-telangiectasia mutated (ATM) kinase and the catalytic subunit of the DNA-dependent protein kinase (DNA-PKcs), regulate cellular responses to DNA double-strand breaks. Increased sensitivity to ionizing radiation (IR) in DNA-PKcs- or ATM-deficient cells emphasizes their important roles in maintaining genome stability. Furthermore, combined knockout of both kinases is synthetically lethal, suggesting functional complementarity. In the current study, using human mammary epithelial cells with ATM levels stably knocked down by >90%, we observed an IR-induced G(2) checkpoint that was only slightly attenuated. In marked contrast, this G(2) checkpoint was significantly attenuated with either DNA-PK inhibitor treatment or RNA interference knockdown of DNA-PKcs, the catalytic subunit of DNA-PK, indicating that DNA-PK contributes to the G(2) checkpoint in these cells. Furthermore, in agreement with the checkpoint attenuation, DNA-PK inhibition in ATM-knockdown cells resulted in reduced signaling of the checkpoint kinase CHK1 as evidenced by reduced CHK1 phosphorylation. Taken together, these results show a DNA-PK-dependent component to the IR-induced G(2) checkpoint, in addition to the well-defined ATM-dependent component. This may have important implications for chemotherapeutic strategies for breast cancers.
authors.affiliation: Environmental Stress and Cancer Group, National Institute of Environmental Health Sciences, NIH, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Sonnet J H
authors.initials: SJ
authors.lastname: Arlander
authors.forename: Bryan T
authors.initials: BT
authors.lastname: Greene
authors.forename: Cynthia L
authors.initials: CL
authors.lastname: Innes
authors.forename: Richard S
authors.initials: RS
authors.lastname: Paules
mesh_terms: D000730:Androstadienes / Q000494:pharmacology; D000903:Antibiotics, Antineoplastic / Q000494:pharmacology; D064007:Ataxia Telangiectasia Mutated Proteins; D001761:Bleomycin / Q000494:pharmacology; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D051747:DNA-Activated Protein Kinase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D019008:Drug Resistance, Neoplasm; D004847:Epithelial Cells / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects; D016195:G2 Phase / Q000187:drug effects / Q000235:genetics / Q000528:radiation effects; D006801:Humans; D042361:Mammary Glands, Human / Q000187:drug effects / Q000201:enzymology / Q000528:radiation effects*; D009154:Mutation; D009687:Nuclear Proteins / Q000037:antagonists & inhibitors / Q000378:metabolism*; D010766:Phosphorylation; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011494:Protein Kinases / Q000187:drug effects / Q000235:genetics; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000378:metabolism; D034741:RNA, Small Interfering / Q000494:pharmacology; D011836:Radiation Tolerance / Q000235:genetics*; D014162:Transfection; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics*; D000077191:Wortmannin
chemical_list: D000730:Androstadienes; D000903:Antibiotics, Antineoplastic; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D047428:Protein Kinase Inhibitors; D034741:RNA, Small Interfering; D025521:Tumor Suppressor Proteins; D001761:Bleomycin; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases; D000077191:Wortmannin