Abstract 5489: Quantitative intracellular immunoassay for monitoring poly (ADP-ribose) in cancer cells treated with PARP inhibitors

Abstract Poly (ADP-ribose) polymerase (PARP) enzymes play a crucial role in cellular DNA repair mechanisms by catalyzing the addition of poly (ADP-ribose) (PAR) chains to target proteins in response to DNA damage. The overactivity of PARP has been implicated in various pathological conditions, including cancer, where PARP inhibitors (PARPi) have emerged as an important therapeutic strategy. Monitoring PAR levels within different cellular environments is essential for understanding PARP activity and optimizing PARP inhibitor efficacy. This study outlines the characterization and development of a sensitive electrochemiluminescence immunoassay designed to quantitatively measure PAR in multiple cancer cell lines treated with PARP inhibitors. The PAR assay was developed on the MSD MULTI-ARRAY platform. Multiple anti-PAR antibodies were tested in all possible capture/detection combinations using a wide range of PAR polymer concentrations. An optimal pair was selected based on response linearity and high sensitivity. Cancer cell lines were subjected to treatment with hydrogen peroxide (MCF-7) or cultured under hypoxia (Cos7, HeLa) over 24 hours, followed by whole cell lysate collection for sample detection using the PAR immunoassay to demonstrate assay utility (e.g., linearity, sample detectability, matrix tolerance). To demonstrate the functional application of the PAR immunoassay, three human cancer cell lines were cultured under standard conditions in 96-well plates and treated with varying concentrations (0.1-100 μM) of three PARP inhibitors over time periods ranging from 1 to 24 hours. Cell lysates were harvested, protein was quantified, and lysates were added to the PAR immunoassay to determine PAR concentration. IC50 curves were generated to evaluate each PARPi’s potency. Following optimization, the PAR immunoassay demonstrated good sensitivity with an LLOD of 3.34 pM in an assay diluent containing protease and phosphatase inhibitors. Using cell lysates from MCF-7, Cos7, or HeLa cells, assay linearity was demonstrated between 0.156-10 µg protein/well, resulting in a linear concentration range of 7 to 2,630 pM. Upon treatment with PARPi, a time and dose-dependent modulation of PAR levels was observed across all cell lines and inhibitors. Specifically, significant changes in IC50 values were found across cell lines and PARP inhibitors tested, indicating the assay’s sensitivity for differentiating inhibitory effects of PARP compounds. This study demonstrates the utility of a novel immunoassay for accurately quantifying PAR in various cancer cell lines subjected to PARP inhibition. This assay offers a robust tool for evaluating PARP inhibitor efficacy and can serve as a valuable asset for researchers in drug development and cellular biology to explore PARP-related cellular responses in different disease contexts. Citation Format: Hans Layman, Dusit Natrakul, Maya Kim, Lars Duerig, Sripriya Ranganathan, Belinda Jackson, Lalitha Srinivasan, Enrique Sanchez-Lemus, Chin-Chi Chen, Caitlin Hansen, Jacob N. Wohlstadter. Quantitative intracellular immunoassay for monitoring poly (ADP-ribose) in cancer cells treated with PARP inhibitors [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5489.