Characterization and Quantification of Poly(ADP-Ribose) in Cells and Tissues by Isotope Dilution Mass Spectrometry

Abstract

Poly(ADP-ribose) (PAR) is a nucleic-acid like biopolymer of various chain lengths with a linear or branched structure which is synthesized by poly(ADP-ribose) polymerases (PARPs). PARPs are involved in various biological processes such as genomic maintenance, cell cycle progression and regulation of cell death. Moreover, PARP inhibitors are currently being evaluated in cancer therapy, either as radio- and chemosensitizers, or as monotherapeutic agents following the concept of synthetic lethality. A detailed analysis of the physiological role of PAR requires both an accurate knowledge of its structure and a reliable method for its quantification. This thesis reports the development of a sensitive, precise and accurate bioanalytical method, based on chromatography-coupled isotope-dilution tandem mass spectrometry, to characterize and quantify PAR with femtomol sensitivity and unequivocal chemical selectivity. To this end, PAR is extracted from cells, purified via solid-phase extraction, and hydrolyzed to specific monomeric units which are then subjected to mass spectrometric analysis. Non-radioactive 13C,15N-labeled PAR was synthesized and used as an authentic chemical standard to account for technical variability during sample preparation and mass spectrometric analysis. Using this method, it is now possible to quantify PAR in different human cancer cell lines, in primary human peripheral blood mononuclear cells (PBMCs), and in mouse tissue both under physiological conditions as well as upon induction of genotoxic stress. This work also demonstrates several potential biological applications of this method. First, LC-MS/MS analysis revealed that DNA-damaging sulfur and nitrogen mustard derivatives robustly induce PARP activation in a dose-dependent manner in a human cancer cell line. Second, ex vivo pharmacodynamic studies were performed in order to define pharmacodynamic properties of clinically relevant PARP inhibitors in freshly isolated PBMCs. Third, poly(ADP-riboyl)ation was analyzed in a spectrum of different mouse tissues which revealed tissue-dependent differences in basal and DNA damage induced PAR levels. Finally a mouse model of systemic inflammation was characterized with regards to its poly(ADP-ribosyl)ation status. In conclusion, this study reports a novel mass spectrometric method for quantifying cellular PAR levels that will be instrumental for in-depth investigations on the metabolism of poly(ADP-ribosyl)ation.