Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Kunzmann, Andrea; Liú, Dan; Annett, Kathryn; Malaisé, M; Thaa, Bastian; Hyland, Paul; Barnett, Yvonne; Bürkle, Alexander

doi:10.1186/1742-4933-3-8

Cited by 24 publications

(11 citation statements)

References 23 publications

Supporting

Mentioning

Contrasting

Unclassified

Order By: Relevance

“…The range in baseline PAR levels measured between all healthy volunteer samples was 39-fold and in patients with cancer was 32-fold, demonstrating a broad heterogeneity inherent in the population. Inter-individual variation in poly(ADP-ribosyl)ation capacity in healthy volunteer PBMCs has been reported previously [19]. While we do not know the reason for the baseline fluctuation in PAR levels measured in healthy volunteers and patients, we are currently conducting flow cytometry and fluorescence microscopy analyses to isolate and identify sensitive subpopulations of PBMCs.…”

Section: Discussionmentioning

confidence: 90%

Modeling Pharmacodynamic Response to the Poly(ADP-Ribose) Polymerase Inhibitor ABT-888 in Human Peripheral Blood Mononuclear Cells

Kinders

Zhang

et al. 2011

PLoS ONE

View full text Add to dashboard Cite

BackgroundPoly(ADP-ribose) polymerase (PARP)facilitates DNA repair and PARP inhibitors may potentiate the effect of DNA-damaging chemotherapeutic agents in patients with cancer. Collection of peripheral blood mononuclear cells (PBMCs)as a surrogate tissue to monitor PARP inhibitor pharmacodynamic effects has several advantages over tumor biopsy collection, including minimally invasive sample collection and the ability to collect multiple samples for longitudinal assessment of drug effect.Methodology/Principal FindingsUsing our previously validated immunoassay for measuring poly(ADP-ribose) (PAR), a product of PARP, in tumor biopsies, we validated a method to quantify PAR levels in PBMCs to monitor the pharmacodynamic effects of the PARP inhibitor ABT-888 in clinical trials. The inter-individual variation in PAR levels was large. No significant difference (P = 0.67) was measured between median baseline PAR levels in 144 healthy volunteers (131.7 pg/1×107 PBMCs [interquartile range, 79.5–241.6]) and 49 patients with cancer (149.2 pg/1×107 PBMCs [interquartile range, 83.2–249.3]). In addition, PAR levels monitored in healthy volunteers over 3 weeks had considerable intra- and inter-individual variation (range, 44–1073 pg PAR/1×107 PBMCs). As a pharmacodynamic model, we quantified changes in PAR levels in human PBMCs treated ex vivo with clinically relevant concentrations of ABT-888. Of 40 healthy volunteer PBMC samples treated with ABT-888, 47.5% had greater than 50% PAR reduction compared to vehicle-treated controls. Considerable inter-sample heterogeneity in PAR levels was measured, and several ABT-888–insensitive samples were identified.Conclusions/SignificanceOur results emphasize the importance of using a validated method to measure PAR levels, and support further investigation into the role of PARP in PBMCs. To this end, the PAR immunoassay has been validated for use with PBMCs and incorporated into clinical trials to assess PBMCs as a potential pharmacodynamic surrogate for tumor biopsies in clinical trials of PARP inhibitors.

show abstract

Section: Discussionmentioning

confidence: 90%

Modeling Pharmacodynamic Response to the Poly(ADP-Ribose) Polymerase Inhibitor ABT-888 in Human Peripheral Blood Mononuclear Cells

Kinders

Zhang

et al. 2011

PLoS ONE

View full text Add to dashboard Cite

show abstract

“…Initially, U-2 OS cells (seeded in a 6-well-plate with 5 × 10 5 cells/well overnight) were treated with 0.1% DMSO (control), 5 µM or 10 µM of regorafenib for 48 h. Cells were harvested, centrifuged and then resuspended in 300 µL of 1 µL substrate solution fluorescein isothiocyanate-Asp(OCH3)-Glu(OCH3)-Val-Asp(OCH3)-fluoromethyl ketone (caspase-3 FITC-DEVD-FMK) or sulforhodamine-Ile-Glu-Thr-Asp-fluoromethyl ketone (caspase-8 Red-IETDFMK) and were incubated at 37 °C under a humidified 5% CO 2 atmosphere for 60 min. Cells were assayed by caspase-3 and caspase-8 stains as described previously [14,16,17].…”

Section: Methodsmentioning

confidence: 99%

Protein Kinase B and Extracellular Signal-Regulated Kinase Inactivation is Associated with Regorafenib-Induced Inhibition of Osteosarcoma Progression In Vitro and In Vivo

Pan

Liu

Hsu

2019

JCM

View full text Add to dashboard Cite

Osteosarcoma is the most common type of bone cancer. Multimodality treatment involving chemotherapy, radiotherapy and surgery is not effective enough to control osteosarcoma. Regorafenib, the oral multi-kinase inhibitor, has been shown to have positive efficacy on disease progression delay in chemotherapy resistant osteosarcoma patients. However anti-cancer effect and mechanism of regorafenib in osteosarcoma is ambiguous. Thus, the aim of this study is to investigate the efficacy and molecular mechanism of regorafenib on osteosarcoma in vitro and in vivo. Human osteosarcomas U-2 OS or MG-63 were treated with regorafenib, miltefosine (protein kinase B (AKT) inhibitor), or PD98059 (mitogen-activated protein/extracellular signal-regulated kinase (MEK) pathway inhibitor) for 24 or 48 h. Cell viability, apoptotic signaling transduction, tumor invasion, expression of tumor progression-associated proteins and tumor growth after regorafenib treatment were assayed by MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide, flow cytometry, transwell assay, Western blotting assay and in vivo animal experiment, respectively. In these studies, we also indicated that regorafenib suppressed cell growth by prompting apoptosis of osteosarcoma cells, which is mediated through inactivation of ERK and AKT signaling pathways. After regorafenib treatment, downregulation of related genes in invasion (vascular endothelial growth factor (VEGF) and matrix metallopeptidase 9 (MMP-9)), proliferation (CyclinD1) and anti-apoptosis (X-linked inhibitor of apoptosis protein (XIAP), myeloid cell leukemia-1 (MCL-1), and cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (C-FLIP)) were found. Moreover, upregulation of caspase-3 and caspase-8 cleavage were also observed. In sum, we suggest that regorafenib has potential to suppress osteosarcoma progression via inactivation of AKT and ERK mediated signaling pathway.

show abstract

“…The positive control of the PAR signal was obtained by incubating cells with 200 µM of etoposide (1 h at 37 °C) (Sigma-Aldrich, Saint Louis, Missouri, USA). Moreover, the negative controls were obtained by incubating cells only with secondary goat anti-mouse 488 IgG (1:530 dilution in PBS) (H+L) (Life Technologies, Rockford, USA) for 1 h at 37 °C; 5000 event files for each sample were acquired individually in a FACS flow cytometer (BD FACSverse, New Jersey, USA) [26] .…”

Section: Experimental Proceduresmentioning

confidence: 99%

Acute telomerase components depletion triggers oxidative stress as an early event previous to telomeric shortening

et al. 2018

View full text Add to dashboard Cite

Loss of function of dyskerin (DKC1), NOP10 and TIN2 are responsible for different inheritance patterns of Dyskeratosis congenita (DC; ORPHA1775). They are key components of telomerase (DKC1 and NOP10) and shelterin (TIN2), and play an important role in telomere homeostasis. They participate in several fundamental cellular processes by contributing to Dyskeratosis congenita through mechanisms that are not fully understood. Presence of oxidative stress was postulated to result from telomerase ablation. However, the resulting disturbed redox status can promote telomere attrition by generating a vicious circle, which promotes cellular senescence. This fact prompted us to study if acute loss of DKC1, NOP10 and TINF2 can promote redox disequilibrium as an early event when telomere shortening has not yet taken place. We generated siRNA-mediated (DKC1, NOP10 and TINF2) cell lines by RNA interference, which was confirmed by mRNA and protein expression analyses. No telomere shortening occurred in any silenced cell line. Depletion of H/ACA ribonucleoproteins DKC1 and NOP10 diminished telomerase activity via TERC down-regulation, and produced alterations in pseudouridylation and ribosomal biogenesis. An increase in the GSSG/GSH ratio, carbonylated proteins and oxidized peroxiredoxin-6 was observed, in addition to MnSOD and TRX1 overexpression in the siRNA DC cells. Likewise, high PARylation levels and high PARP1 protein expression were detected. In contrast, the silenced TINF2 cells did not alter any evaluated oxidative stress marker. Altogether these findings lead us to conclude that loss of DKC1 and NOP10 functions induces oxidative stress in a telomere shortening independent manner.

show abstract

Flow-cytometric assessment of cellular poly(ADP-ribosyl)ation capacity in peripheral blood lymphocytes

Cited by 24 publications

References 23 publications

Modeling Pharmacodynamic Response to the Poly(ADP-Ribose) Polymerase Inhibitor ABT-888 in Human Peripheral Blood Mononuclear Cells

Modeling Pharmacodynamic Response to the Poly(ADP-Ribose) Polymerase Inhibitor ABT-888 in Human Peripheral Blood Mononuclear Cells

Protein Kinase B and Extracellular Signal-Regulated Kinase Inactivation is Associated with Regorafenib-Induced Inhibition of Osteosarcoma Progression In Vitro and In Vivo

Acute telomerase components depletion triggers oxidative stress as an early event previous to telomeric shortening

Contact Info

Product

Resources

About