title: Poly(ADP-ribose) polymerase activity prevents signaling pathways for cell cycle arrest after DNA methylating agent exposure.
abstract: Mouse fibroblasts, deficient in DNA polymerase beta, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase beta null cells are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8-naphthalimide (4-AN), an inhibitor of poly(ADP-ribose) polymerase (PARP) activity. Experiments with synchronized cells suggest that exposure during S-phase of the cell cycle is required for the 4-AN effect. 4-AN elicits a similar extreme sensitization to the thymidine analog, 5-hydroxymethyl-2'-deoxyuridine, implicating the requirement for an intermediate of DNA repair. In PARP-1-expressing fibroblasts treated with a combination of MMS and 4-AN, a complete inhibition of DNA synthesis is apparent after 4 h, and by 24 h, all cells are arrested in S-phase of the cell cycle. Continuous incubation with 4-AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM (ataxia telangiectasia-mutated) and ATR (ATM and Rad3-related), has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, the addition of caffeine leads to arrest of cells in G(2)/M rather than S-phase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 after treatment with MMS and 4-AN, which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through maintenance of an ATR and Chk1-dependent S-phase checkpoint.
authors.affiliation: Laboratory of Structural Biology, NIEHS, National Institutes of Health, Research Triangle Park, North Carolina 27709, USA.
authors.forename: Julie K
authors.initials: JK
authors.lastname: Horton
authors.forename: Donna F
authors.initials: DF
authors.lastname: Stefanick
authors.forename: Jana M
authors.initials: JM
authors.lastname: Naron
authors.forename: Padmini S
authors.initials: PS
authors.lastname: Kedar
authors.forename: Samuel H
authors.initials: SH
authors.lastname: Wilson
mesh_terms: D015057:1-Naphthylamine / Q000031:analogs & derivatives* / Q000494:pharmacology; D000818:Animals; D018906:Antineoplastic Agents, Alkylating / Q000494:pharmacology; D002453:Cell Cycle / Q000187:drug effects / Q000502:physiology*; D004247:DNA / Q000378:metabolism*; D004249:DNA Damage / Q000187:drug effects; D019175:DNA Methylation; D005347:Fibroblasts / Q000187:drug effects / Q000378:metabolism; D008741:Methyl Methanesulfonate / Q000494:pharmacology; D051379:Mice; D053644:Naphthalimides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D011065:Poly(ADP-ribose) Polymerases / Q000235:genetics / Q000378:metabolism*; D015363:Quinolones / Q000494:pharmacology; D015398:Signal Transduction / Q000502:physiology*
chemical_list: D018906:Antineoplastic Agents, Alkylating; D053644:Naphthalimides; D000067856:Poly(ADP-ribose) Polymerase Inhibitors; D015363:Quinolones; C086538:4-amino-1,8-naphthalimide; D004247:DNA; D015057:1-Naphthylamine; D008741:Methyl Methanesulfonate; D011065:Poly(ADP-ribose) Polymerases