Enhanced DNA-PK-mediated RPA2 hyperphosphorylation in DNA polymerase eta-deficient human cells treated with cisplatin and oxaliplatin.
The chemotherapeutic drugs cisplatin and oxaliplatin act by induction of DNA damage, including monoadducts, intrastrand and interstrand crosslinks. An increased understanding of the repair and replication of platinum-damaged DNA is required to improve the effectiveness of these drugs in killing cancer cells. We have investigated the effect of expression of DNA polymerase eta (poleta), a translesion synthesis (TLS) enzyme, on the response of human cell lines to cisplatin and oxaliplatin. Poleta-deficient cells are more sensitive to both drugs than are normal cells. In poleta-deficient cells, drug treatment leads to prolonged S-phase arrest, and increased phosphorylation of the phosphatidylinositol-3-kinase-related protein kinase (PIKK) substrates Chk1, p95/Nbs1 and RPA2, the 34kDa subunit of replication protein A. Cisplatin- and oxaliplatin-induced hyperphosphorylation of RPA2, and association of the hyperphosphorylated protein with chromatin, is elevated in poleta-deficient cells. Cisplatin-induced phosphorylation of RPA2 on serine 4/serine 8, but not on serine 33, is inhibited by the DNA-PK inhibitor, NU7441, but not by the ATM inhibitor, KU-55933. Cisplatin-induced DNA-PK-dependent hyperphosphorylation of RPA2 on serine 4/serine 8 occurs after recruitment of RPA to chromatin, as determined by immunofluorescence and by subcellular fractionation. ATR is required both for recruitment of RPA2 to chromatin and its subsequent hyperphosphorylation on serine 4/serine 8 by DNA-PK, since CGK733, an inhibitor of ATM and ATR, blocked both recruitment and hyperphosphorylation. Thus, increased sensitivity to cisplatin and oxaliplatin in DNA poleta-deficient cells is associated with prolonged S-phase arrest, and enhanced PIKK-signalling, in particular activation of DNA-PK-dependent hyperphosphorylation of RPA2 on serines 4 and 8.
Cruet-Hennequart
Séverine
S
DNA Damage Response Laboratory, Department of Biochemistry, and National Centre for Biomedical Engineering Science, National University of Ireland, Galway, Galway City, Ireland.
Glynn
Macdara T
MT
Murillo
Laura S
LS
Coyne
Seamus
S
Carty
Michael P
MP
D000970:Antineoplastic Agents / Q000494:pharmacology*; D059447:Cell Cycle Checkpoints / Q000187:drug effects; D018797:Cell Cycle Proteins / Q000378:metabolism; D002460:Cell Line; D000071877:Checkpoint Kinase 1; D002843:Chromatin / Q000187:drug effects / Q000378:metabolism; D002945:Cisplatin / Q000494:pharmacology*; D004259:DNA-Directed DNA Polymerase / Q000235:genetics*; D006801:Humans; D009154:Mutation; D009687:Nuclear Proteins / Q000378:metabolism; D009944:Organoplatinum Compounds / Q000494:pharmacology*; D000077150:Oxaliplatin; D010766:Phosphorylation; D011494:Protein Kinases / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D051716:Replication Protein A / Q000235:genetics / Q000378:metabolism*; D000099270:Y-Family DNA Polymerases
D000970:Antineoplastic Agents; D018797:Cell Cycle Proteins; D000071877:Checkpoint Kinase 1; D002843:Chromatin; D002945:Cisplatin; D004259:DNA-Directed DNA Polymerase; D009687:Nuclear Proteins; D009944:Organoplatinum Compounds; D000077150:Oxaliplatin; D011494:Protein Kinases; D051716:Replication Protein A; C112156:NBN protein, human; C000605867:CHEK1 protein, human; C516794:RPA2 protein, human; C112338:Rad30 protein; D000099270:Y-Family DNA Polymerases