Enhanced DNA Accessibility and Increased DNA Damage Induced by the Absence of Poly(ADP-ribose) Hydrolysis

Abstract

Poly(ADP-ribose) (PAR) is a therapeutic target primarily identified through inhibiting its synthesis by PAR polymerase-1 (PARP-1). However, inhibiting its hydrolysis by PAR glycohydrolase (PARG) has therapeutic potential in cancer. Unknown is the effect of elevated PAR levels on cellular processes and if this effect can enhance the therapeutic value of PARG. Here, we demonstrate in PARG null embryonic trophoblast stem (TS) cells that the absence of PAR hydrolysis led to PAR-modified histones H1, H2A, and H2B. To determine if this led to the differential vulnerability of DNA to modification, TS cells were treated with DNA-modifying agents. The results demonstrate increased DNA laddering by micrococcal nuclease and an increased amount of DNA intercalation by acridine orange in PARG null-TS cells. This increased access to PARG null-TS cell DNA was further verified by the detection of increased DNA damage following treatment with UV radiation and a minimal dose of the DNA-alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine. Further, this DNA damage was predominantly unrepaired 12 h after treatment in PARG null-TS cells. Finally, TS cells were treated with DNA-modifying chemotherapeutic agents. The results demonstrate up to 4-fold increases in cell death in PARG null-TS cells after treatment with epirubicin or sub-IC(50) doses of cisplatin and cyclophosphamide. Taken together, we provide compelling evidence that increased DNA access induced by the absence of PARG enhances the efficacy of DNA-modifying agents. Thus, this study demonstrates that greater DNA accessibility, increased DNA damage, and increased cell death all contribute to the PARG null cell phenotype in response to genotoxic stress.