Abstract 1270: Elevated levels of poly(ADP-ribose) lead to novel apoptotic signaling and the induction of caspase-independent cell death

Abstract

Abstract The metabolism of poly(ADP-ribose) (PAR) is activated by DNA damage, where PAR polymerase-1 (PARP-1) transfers ADP-ribose onto nuclear proteins to form PAR and PAR glycohydrolase (PARG) catalyzes its degradation. Previous findings show that high levels of PAR lead to its presence in the cytoplasm, where it signals the release of the proapoptotic protein, apoptosis inducing factor (AIF). Our studies suggest that inhibiting PAR glycohydrolase (PARG), and thus promoting increased levels of PAR, will increase the efficacy of anti-cancer agents by promoting this alternative cell death pathway mediated by AIF. The objective of this study is to elucidate the pathway of cytoplasm PAR cell death signaling in cancer cells. We utilized MDA-MB-231 breast cancer cells to reveal the ability of AIF to induce cancer cell death following caspase inhibition. In addition, PARG null embryonic trophoblast stem cells were utilized to determine the cytoplasmic PAR signaling pattern and to purify the PAR-modified nuclear proteins released in the cytoplasm. FACS analysis demonstrated equivalent levels of cell death with or without caspases and Western blot revealed a robust release of AIF from the mitochondria following DNA damage. Following low-dose DNA-damaging agent treatment in PARG null cells, cytoplasmic PAR was detected concurrently with AIF translocation to the nucleus, as shown by Western blot and confocal immunofluorescence. In PARG null cells in the absence of stress, cytoplasmic PAR levels were maximal after 3 days as shown by cell fractionations and Western blot. The cytoplasmic fraction containing PAR was then purified using dihydroxyboronyl Bio-Rex affinity chromatography. The purified PAR-modified proteins were treated with phosphodiesterase to remove all PAR and SDS-PAGE analysis demonstrated several proteins ranging from 20-50 kDa, which together serve as the cytoplasmic signal that causes AIF release from the mitochondria. These proteins potentially reveal novel nuclear-to-mitochondrial cell signaling ligands that are necessary for the induction of the AIF pathway of caspase-independent cell death. Taken together, the results demonstrate the importance of AIF in cancer cell death and novel insight into the cell death signaling by PAR that produces AIF-mediated cell death. Therefore, ability to increase PAR levels by inhibiting PARG is potentially a unique method to induce the death of cancer cells by an alternative cell death pathway. This research was supported by funding from the American Cancer Society and the Pharmaceutical Research Manufacturers of America. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1270.