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

Mouse fibroblasts, deficient in DNA polymerase ß (ß-pol), are hypersensitive to monofunctional DNA methylating agents such as methyl methanesulfonate (MMS). Both wild-type, and in particular repair deficient ßpol null cells, are highly sensitized to the cytotoxic effects of MMS by 4-amino-1,8naphthalimide (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-1expressing 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 4AN is required to maintain the cell cycle arrest. Caffeine, an inhibitor of the upstream checkpoint kinases ATM and ATR, has no effect on the early inhibition of DNA synthesis, but cells are no longer able to maintain the block after 8 h. Instead, addition of caffeine leads to arrest of cells in G2/M rather than Sphase after 24 h. Analysis of signaling pathways in cell extracts reveals an activation of Chk1 following treament with MMS and 4AN which can be suppressed by caffeine. Our results suggest that inhibition of PARP activity results in sensitization to MMS through activation of an ATR and Chk1-dependent Sphase checkpoint.