Abstract #3019: Validated sensitive immunological determination of PARP-1 protein levels and activity in cultured cells and human lymphocytes
Authors: Evan Mulligan, Tomasz Zaremba, Yvette Drew, Zdenek Hostomsky, Elizabeth Plummer, Nicola Curtin
Journal: Cancer Research (2009)

Background:  PARP-1 is a nuclear enzyme that signals DNA strand breaks for repair. There is considerable interest in PARP-1 in health and disease and the development of PARP inhibitors for cancer therapy. A number of PARP inhibitors have now entered clinical trials. We report here the development of sensitive assays for both PARP-1 protein and activity, validated to GCLP, that are applicable to pre-clinical and clinical samples that may be used to guide pre-clinical development of PARP inhibitors and evaluation of their activity in clinical trials.  Methods:  We determined PARP-1 protein expression and activity in fresh cultured cells or human lymphocytes or following freezing in culture medium supplemented with 10% DMSO and protease inhibitors. PARP-1 protein was measured by semi-quantitative Western blot using anti PARP-1 C2-10 primary and polyclonal Goat antimouse secondary antibody (Ab) by reference to 40-2.5ng PARP-1 protein standard curve and K562 cell lysates as QC samples. Maximum stimulatable PARP activity was determined following incubation of permeabilised cells with NAD and 30-mer oligonucleotide by immunological detection of ADP-ribose polymers (PAR) (1) following blotting on to hybond-N membrane using a purpose-built (PE 300: Polyethylene) manifold with 12mm radii wells using 10H primary and a Goat anti-mouse secondary Ab by reference to a PAR standard curve and cultured cell QC samples. Both protein and activity was quantified by ECL chemiluminescence.  Results:  The limit of detection of PARP-1 protein and PARP-1 activity was 2.5 ng and 0.02 pmol, respectively. Inter assay variability was 40 % coefficient of variation (CV) for protein and 25% CV for activity but intra assay variability was below 15% CV for both assays. For this reason we recommend that all samples from one patient are run in the same assay. PARP activity could be detected in as few as 5000 cultured human cancer cells and 10 000 human lymphocytes and only 30µg soluble protein extract from cultured cells and human lymphocytes were needed to measure PARP-1 protein levels. Thus both protein and activity could be measured in triplicate samples in 2 independent assays with only 10 ml blood. PARP activity, and its inhibition by AG014699, was maintained for at least 14 weeks at -80 o C. This assay has been used as a secondary endpoint in clinical trials of the Pfizer PARP inhibitor AG-014699.  Conclusions:  Determination of PARP activity and expression can be determined in cultured cells and patient lymphocytes. Rigorous guidelines for the preparation of samples alongside standards and quality controls provide a robust and highly sensitive assay for evaluation of novel PARP inhibitors pre-clinically and clinically.     1-Plummer ER et al   Clinical Cancer Research.   11  3402-3409 (2005)     Citation Information:  In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 3019.