title: Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts.
abstract: Treatment of base excision repair-proficient mouse fibroblasts with the DNA alkylating agent methyl methanesulfonate (MMS) and a small molecule inhibitor of PARP-1 results in a striking cell killing phenotype, as previously reported. Earlier studies showed that the mechanism of cell death is apoptosis and requires DNA replication, expression of PARP-1, and an intact S-phase checkpoint cell signaling system. It is proposed that activity-inhibited PARP-1 becomes immobilized at DNA repair intermediates, and that this blocks DNA repair and interferes with DNA replication, eventually promoting an S-phase checkpoint and G(2)-M block. Here we report studies designed to evaluate the prediction that inhibited PARP-1 remains DNA associated in cells undergoing repair of alkylation-induced damage. Using chromatin immunoprecipitation with anti-PARP-1 antibody and qPCR for DNA quantification, a higher level of DNA was found associated with PARP-1 in cells treated with MMS plus PARP inhibitor than in cells without inhibitor treatment. These results have implications for explaining the extreme hypersensitivity phenotype after combination treatment with MMS and a PARP inhibitor.
authors.affiliation: Laboratory of Structural Biology, National Institute of Environmental Health Sciences, NIH, 111 T.W. Alexander Drive, Research Triangle Park, NC 27709, USA.
authors.forename: Padmini S
authors.initials: PS
authors.lastname: Kedar
authors.forename: Donna F
authors.initials: DF
authors.lastname: Stefanick
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D000478:Alkylation / Q000187:drug effects; D000818:Animals; D002843:Chromatin / Q000378:metabolism; D047369:Chromatin Immunoprecipitation; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage; D004791:Enzyme Inhibitors / Q000494:pharmacology; D051379:Mice; D008954:Models, Biological; D009687:Nuclear Proteins / Q000378:metabolism; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000378:metabolism*; D011485:Protein Binding / Q000187:drug effects; D015203:Reproducibility of Results; D012995:Solubility / Q000187:drug effects; D013347:Subcellular Fractions / Q000187:drug effects / Q000378:metabolism
chemical_list: D002843:Chromatin; D004791:Enzyme Inhibitors; D009687:Nuclear Proteins; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D004247:DNA; D011065:Poly(ADP-ribose) Polymerases