Predicting enhanced cell killing through PARP inhibition

Abstract

PARP inhibitors show promise as combination and single agents in cancer chemotherapy. Here, we evaluate results obtained with mouse fibroblasts and the common laboratory PARP inhibitor 4-amino-1,8-naphthalimide (4-AN) and analyze the potential for enhanced cytotoxicity following the combination of a DNA-damaging agent and a PARP inhibitor. Methylated DNA bases are repaired by the monofunctional glycosylase-initiated single-nucleotide base excision repair (BER) pathway. An intermediate of this process has a single-nucleotide gap in double-stranded DNA containing the 5'-deoxyribose phosphate (dRP) group at one margin. This 5'-dRP group is removed by the lyase activity of pol β prior to gap filling; then completion of repair is by DNA ligation. PARP-1 binds to and is activated by the 5'-dRP group-containing intermediate, and poly(ADP-ribos)ylation is important for efficient repair. 4-AN-mediated sensitization to the methylating chemotherapeutic agent temozolomide is extreme, producing a level of cytotoxicity not seen with either agent alone. In contrast, with agents producing oxidative DNA damage repaired by bifunctional glycosylase-initiated BER, there is only weak sensitization by cotreatment with PARP inhibitor. Other clinically used DNA-damaging agents repaired by different DNA repair pathways also reveal minimal 4-AN-mediated sensitization. This information has potentially important implications for strategic use of PARP inhibitors in chemotherapy.

Figure 1

A, Scheme of pol β-dependent BER initiated by a monofunctional (left side) or bifunctional (right side) DNA glycosylase showing formation of 5′-or 3′-blocking groups (circled), respectively, in repair intermediates. The specific interaction between PARP-1 and the5′-dRP-containing intermediate is indicated. B, Inhibitory effect of 4-AN (10 μM) on PAR synthesis in MMS-treated MEFs (10 mM for 20 min at 4 °C) as measured by a commercial ELISA assay (Trevigen). C, Sensitivity of wild-type MEFs to a 1 h exposure to MMS (circles) and the sensitization obtained by co-treatment with 4-AN (10 μM for 24 h; squares). D, Sensitivity of wild-type mouse fibroblasts to a 1 h exposure to peroxynitrite (circles) and the absence of sensitization provided by co-treatment with 4-AN(10 μM for 24 h; squares). The plot was drawn to the same scale as in C. Cell sensitivity was obtained by growth inhibition assays as described previously (12). E, Comparison of 4-AN-mediated fold-sensitization of wild-type MEFs to MMS (40-fold) and peroxynitrite (negligible).

Figure 2

A, Hypersensitivity (3.2-fold) of pol β null mouse fibroblasts (open symbols) to TMZ (4 h exposure). B, Sensitization to TMZ cytotoxicity by 24 h exposure to the PARP inhibitor, 4-AN (squares). C, Comparison of 4-AN mediated sensitization in wild-type and pol β null MEFs to agents resulting in damage repaired by monofunctional and bifunctional BER pathways. D, Low level sensitization by 4-AN of pol β null cells to peroxynitrite.

Figure 3

Level of hypersensitivity of pol β null MEFs (open circles) to exposure to A, cisPt (1 h); B, Ara C (24 h) or C, CPT (24 h) compared with wild-type MEFs (closed circles). A–C, Sensitization of pol β null (open squares) and wild-type MEFs (closed squares) by 24 h exposure to 4-AN (10 μM). D, Comparison of 4-AN mediated sensitization to TMZ and other types of clinically-utilized DNA damaging agents in wild-type and pol β null mouse fibroblasts.