Increased PARP-1 association with DNA in alkylation damaged, PARP-inhibited mouse fibroblasts

• Published in: Molecular cancer research 2012-03, Vol.10 (3), p.360-368
• Main Authors: Kedar, Padmini S, Stefanick, Donna F, Horton, Julie K, Wilson, Samuel H
• Format: Article
• Language: English
• Subjects: Alkylation - drug effects; Animals; Chromatin - metabolism; Chromatin Immunoprecipitation; DNA - metabolism; DNA Damage; Enzyme Inhibitors - pharmacology; Mice; Models, Biological; Nuclear Proteins - metabolism; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases - metabolism; Protein Binding - drug effects; Reproducibility of Results; Solubility - drug effects; Subcellular Fractions - drug effects; Subcellular Fractions - metabolism
• ISSN: 1541-7786; 1557-3125
• DOI: 10.1158/1541-7786.MCR-11-0477

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.