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.

Figure 1

Working model for studies of the effects of PARP inhibition during BER of alkylated DNA bases in replicating cells. After removal of the MMS-induced alkylated base by a monofunctional DNA glycosylase, AP endonuclease (APE1) incises the abasic site leaving a gap with the 5′-deoxyribose phosphate group (dRP) at the margin. In the normal base excision repair (BER) pathway depicted on the left, many BER factors are in a preformed complex that recognizes the BER intermediate shown at the top. Poly(ADP ribose) or PAR-adducted PARP-1 is depicted as releasing from the BER complex during repair. In the presence of the PARP inhibitor 4-AN depicted on the right, inhibited PARP-1 remains bound to the BER intermediate with the 5′ -dRP group. This eventually leads to replication fork stalling, checkpoint activation and apoptosis.

Figure 2

Mouse fibroblast synchronization, treatment and characterization. All experiments were conducted as described under Methods. A, Synchronization and treatment protocol. Mouse fibroblasts in log phase were grown for 48 h with medium containing only 0.2% FBS (serum withdrawal), followed by culture with 2.5 µM APH in DMEM with 10% FBS for 16 h. Cells were released from the APH block by washing then addition of DMEM containing 10% FBS. After 4 h, the cells were treated with 0.25 mM MMS or 10 µM 4-AN or the combination in complete medium for 1 h. The medium was removed and the cells were cultured for 1 h (repair) +/− 4-AN in complete medium. Control cells were mock-treated in parallel at each step. B, Assessment of PARP-1 protein level in the cell extracts as a function of treatment. After treatment as illustrated in Panel A, cell extracts were immunoblotted with anti-PARP-1 monoclonal antibody as described; the membrane was stripped and probed with monoclonal antibody against G3PDH, used as loading control.

Figure 3

DNA associated with PARP in ChIP samples from control and treated cells. Experiments were conducted as described under Methods. DNA was purified from the various ChIP samples and measured by qPCR using A, isochore primers and B, DNA polymerase β primers. The amount of DNA in each type of ChIP sample is represented as the qPCR signal relative to a constant amount of input chromatin DNA. The data represent average values (with S.D. bars) from 3 independent ChIP samples, each one measured in triplicate. In panels A and B, the triple asterisk symbols indicate difference from the respective control samples at p < 0.001 using the two-sample t-test, and in panel B the single asterisk symbols indicate difference from the control samples at p < 0.05 using the two-sample t-test. The results with the MMS + 4-AN samples in panels A and B are different from all other samples at p < 0.001 using the two-sample t-test.

Figure 4

Analysis of PARP-1 protein in the nuclear fraction isolated from cells after the various cell treatments. Experiments were conducted as described under Methods. A, The nuclear soluble protein fraction and chromatin-associated protein fraction obtained from cells, treated as indicated, by a biochemical fractionation procedure were analyzed by immunoblotting with monoclonal antibody against PARP-1 and polyclonal antibody against histone H3 or Lamin A, as a control. Lanes 1 and 5, control cells; lanes 2 and 6, MMS treated cells; lanes 3 and 7, 4-AN treated cells; lanes 4 and 8, MMS + 4-AN treated cells. B, Increasing amounts of chromatin-associated protein fraction (lanes 1 and 5, 25 µg; lanes 2 and 6, 50 µg; lanes 3 and 7, 75 µg; lanes 4 and 8, 100 µg) from control and MMS + 4-AN-treated cells were analyzed for PARP-1 by immunoblotting. The solid symbols at the top of the immunoblot indicate increasing amounts of protein added. Histone H3 was probed as an internal loading control. C, Quantification of PARP-1 in the chromatin-associated protein fraction from control cells (circles) or cells treated with the MMS + 4-AN combination (squares) shown in panel B. The lines, solid and dashed, represent a curve fitting of the respective data points.

Figure 5

Co-immunoprecipitation analysis of proteins in ChIP samples prepared from cells treated with MMS + 4-AN. Experiments were conducted as described under Methods and as illustrated in Figure 2. Two types of ChIP samples were prepared after treatment of cells as described; one with the anti-PARP-1 antibody, as usual; and the other with negative control non-immune IgG. For immunoblotting analysis (IB), proteins in the two ChIP samples were separated by 4–12% SDS-PAGE and transferred to a membrane. Then, the membrane was probed with antibodies against PARP-1, NBS1, MRE11, RPA70, and ATR, as illustrated. IB probing with non-immune IgG was negative corresponding to the each of these proteins (not shown).