Poly(ADP-ribose) Polymerase Activity Prevents Signaling Pathways for Cell Cycle Arrest after DNA Methylating Agent Exposure

• Published in: The Journal of biological chemistry 2005-04, Vol.280 (16), p.15773-15785
• Main Authors: Horton, Julie K., Stefanick, Donna F., Naron, Jana M., Kedar, Padmini S., Wilson, Samuel H.
• Format: Article
• Language: English
• Subjects: 1-Naphthylamine - analogs & derivatives; 1-Naphthylamine - pharmacology; Animals; Antineoplastic Agents, Alkylating - pharmacology; Cell Cycle - drug effects; Cell Cycle - physiology; DNA - metabolism; DNA Damage - drug effects; DNA Methylation; Fibroblasts - drug effects; Fibroblasts - metabolism; Methyl Methanesulfonate - pharmacology; Mice; Naphthalimides; Poly(ADP-ribose) Polymerase Inhibitors; Poly(ADP-ribose) Polymerases - genetics; Poly(ADP-ribose) Polymerases - metabolism; Quinolones - pharmacology; Signal Transduction - physiology
• ISSN: 0021-9258; 1083-351X
• DOI: 10.1074/jbc.M413841200

Mouse fibroblasts, deficient in DNA polymerase β, are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type and, in particular, repair-deficient DNA polymerase β 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 G2/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.