Involvement of the ATR- and ATM-dependent checkpoint responses in cell cycle arrest evoked by pierisin-1.
Pierisin-1 identified from the cabbage butterfly, Pieris rapae, is a novel mono-ADP-ribosylating toxin that transfers the ADP-ribose moiety of NAD at N(2) of dG in DNA. Resulting mono-ADP-ribosylated DNA adducts cause mutations and the induction of apoptosis. However, little is known about checkpoint responses elicited in mammalian cells by the formation of such bulky DNA adducts. In the present study, it was shown that DNA polymerases were blocked at the specific site of mono-ADP-ribosylated dG, which might lead to the replication stress. Pierisin-1 treatment of HeLa cells was found to induce an intra-S-phase arrest through both ataxia telangiectasia mutated (ATM) and Rad3-related (ATR) and ATM pathways, and ATR pathway also contributes to a G(2)-M-phase delay. In the colony survival assays, Rad17(-/-) DT40 cells showed greater sensitivity to pierisin-1-induced cytotoxicity than wild-type and ATM(-/-) DT40 cells, possibly due to defects of checkpoint responses, such as the Chk1 activation. Furthermore, apoptotic 50-kb DNA fragmentation was observed in the HeLa cells, which was well correlated with occurrence of phosphorylation of Chk2. These results thus suggest that pierisin-1 treatment primarily activates ATR pathway and eventually activates ATM pathway as a result of the induction of apoptosis. From these findings, it is suggested that mono-ADP-ribosylation of DNA causes a specific type of fork blockage that induces checkpoint activation and signaling.
Shiotani
Bunsyo
B
Cancer Prevention Basic Research Project, National Cancer Center Research Institute, 5-1-1 Tsukiji, Chuo-ku, Tokyo 104-0045, Japan.
Kobayashi
Masahiko
M
Watanabe
Masahiko
M
Yamamoto
Ken-Ichi
K
Sugimura
Takashi
T
Wakabayashi
Keiji
K
D036002:ADP Ribose Transferases; D000246:Adenosine Diphosphate Ribose / Q000378:metabolism*; D000818:Animals; D001260:Ataxia Telangiectasia; D064007:Ataxia Telangiectasia Mutated Proteins; D015153:Blotting, Western; D002080:Butterflies / Q000378:metabolism; D002453:Cell Cycle / Q000187:drug effects*; D018797:Cell Cycle Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D000071877:Checkpoint Kinase 1; D064447:Checkpoint Kinase 2; D003114:Colony-Forming Units Assay; D004247:DNA / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*; D004789:Enzyme Activation; D005347:Fibroblasts / Q000378:metabolism; D016147:Genes, Tumor Suppressor; D006367:HeLa Cells / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D047468:Immunoprecipitation; D019476:Insect Proteins / Q000494:pharmacology*; D051379:Mice; D018345:Mice, Knockout; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D017346:Protein Serine-Threonine Kinases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism* / Q000502:physiology*; D034741:RNA, Small Interfering / Q000494:pharmacology; D015398:Signal Transduction; D025521:Tumor Suppressor Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000502:physiology*
D018797:Cell Cycle Proteins; D004268:DNA-Binding Proteins; D019476:Insect Proteins; D034741:RNA, Small Interfering; C113575:Rad17 protein, human; D025521:Tumor Suppressor Proteins; C113557:pierisin protein, insect; D000246:Adenosine Diphosphate Ribose; D004247:DNA; D036002:ADP Ribose Transferases; D011494:Protein Kinases; D064447:Checkpoint Kinase 2; C576324:ATM protein, human; C576597:ATR protein, human; D064007:Ataxia Telangiectasia Mutated Proteins; C576325:Atm protein, mouse; C000605867:CHEK1 protein, human; C578634:CHEK2 protein, human; D000071877:Checkpoint Kinase 1; C000605869:Chek1 protein, mouse; C578633:Chek2 protein, mouse; D017346:Protein Serine-Threonine Kinases