title: Rad9, Rad17, TopBP1 and claspin play essential roles in heat-induced activation of ATR kinase and heat tolerance.
abstract: Hyperthermia is widely used to treat patients with cancer, especially in combination with other treatments such as radiation therapy. Heat treatment per se activates DNA damage responses mediated by the ATR-Chk1 and ATM-Chk2 pathways but it is not fully understood how these DNA damage responses are activated and affect heat tolerance. By performing a genetic analysis of human HeLa cells and chicken B lymphoma DT40 cells, we found that heat-induced Chk1 Ser345 phosphorylation by ATR was largely dependent on Rad9, Rad17, TopBP1 and Claspin. Activation of the ATR-Chk1 pathway by heat, however, was not associated with FancD2 monoubiquitination or RPA32 phosphorylation, which are known as downstream events of ATR kinase activation when replication forks are stalled. Downregulation of ATR, Rad9, Rad17, TopBP1 or Claspin drastically reduced clonogenic cell viability upon hyperthermia, while gene knockout or inhibition of ATM kinase reduced clonogenic viability only modestly. Suppression of the ATR-Chk1 pathway activation enhanced heat-induced phosphorylation of Chk2 Thr68 and simultaneous inhibition of ATR and ATM kinases rendered severe heat cytotoxicity. These data indicate that essential factors for activation of the ATR-Chk1 pathway at stalled replication forks are also required for heat-induced activation of ATR kinase, which predominantly contributes to heat tolerance in a non-overlapping manner with ATM kinase.
authors: Department of Molecular Oncology, Graduate School of Medical Sciences, Kyushu University, Fukuoka, Japan.
authors: Munkhbold
authors: M
authors: Tuul
authors: Hiroyuki
authors: H
authors: Kitao
authors: Makoto
authors: M
authors: Iimori
authors: Kazuaki
authors: K
authors: Matsuoka
authors: Shinichi
authors: S
authors: Kiyonari
authors: Hiroshi
authors: H
authors: Saeki
authors: Eiji
authors: E
authors: Oki
authors: Masaru
authors: M
authors: Morita
authors: Yoshihiko
authors: Y
authors: Maehara
mesh_terms: D000064:Acclimatization / Q000187:drug effects*; D048868:Adaptor Proteins, Signal Transducing / Q000494:pharmacology*; D000818:Animals; D064007:Ataxia Telangiectasia Mutated Proteins; D002110:Caffeine / Q000494:pharmacology; D002352:Carrier Proteins / Q000494:pharmacology*; D018797:Cell Cycle Proteins / Q000172:deficiency / Q000378:metabolism* / Q000494:pharmacology*; D045744:Cell Line, Tumor; D000071877:Checkpoint Kinase 1; D002645:Chickens; D004249:DNA Damage; D004268:DNA-Binding Proteins / Q000494:pharmacology*; D004789:Enzyme Activation / Q000187:drug effects*; D005455:Fluorescent Antibody Technique; D005786:Gene Expression Regulation / Q000502:physiology; D006367:HeLa Cells; D006358:Hot Temperature; D006801:Humans; D009687:Nuclear Proteins / Q000494:pharmacology*; D010766:Phosphorylation / Q000187:drug effects; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism*; D034622:RNA Interference; D034741:RNA, Small Interfering
chemical_list: D048868:Adaptor Proteins, Signal Transducing; C488158:CLSPN protein, human; D002352:Carrier Proteins; D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D034741:RNA, Small Interfering; C113575:Rad17 protein, human; C110513:TOPBP1 protein, human; C069930:rad9 protein; D002110:Caffeine; D011494:Protein Kinases; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C000605867:CHEK1 protein, human; D000071877:Checkpoint Kinase 1; D017346:Protein Serine-Threonine Kinases
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