학술논문
Performance of a phosphoflow assay to determine phosphorylation of S6 ribosomal protein as a pharmacodynamic read out for mTOR inhibition.
Document Type
Academic Journal
Author
Abdel-Kahaar E; Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: e.ahmed@klinikum-stuttgart.de.; Kabakchiev M; Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: m.kabakchiev@klinikum-stuttgart.de.; Hartmann B; MVZ Waiblingen, Praxis für Nieren- und Hochdruckerkrankungen, Beinsteiner Strasse 8/3, D-71334 Waiblingen, Germany. Electronic address: Hartmann.Bertram@mvz-gmbh.de.; Wieland E; Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: e.wieland@klinikum-stuttgart.de.; Shipkova M; Central Institute for Clinical Chemistry and Laboratory Medicine, Klinikum Stuttgart, Kriegsbergstrasse 62, D-70174 Stuttgart, Germany. Electronic address: m.shipkova@klinikum-stuttgart.de.
Source
Publisher: Elsevier Science Country of Publication: United States NLM ID: 0133660 Publication Model: Print-Electronic Cited Medium: Internet ISSN: 1873-2933 (Electronic) Linking ISSN: 00099120 NLM ISO Abbreviation: Clin Biochem Subsets: MEDLINE
Subject
Language
English
Abstract
Objectives: The S6 ribosomal protein (S6RP) is phosphorylated by the mammalian target of rapamycin (mTOR). The objective of this study was to assess the analytical suitability of a commercial kit-based phosphoflow cytometry protocol using whole blood (WBS) to measure the level of phosphorylated S6RP (p-S6RP) in T-cell subsets to study the pharmacodynamic effects of mTOR inhibitors (mTORi).
Design and Methods: A kit was used for fixation and permeabilization of mitogen-stimulated cells, and p-S6RP was assessed separately in CD3+CD4+ and CD3+CD8+ cells by employing an anti-phospho-Ser235/236 antibody. Specificity, linearity, within-run precision and stability were investigated in either WBS spiked with everolimus and non-mTORi immunosuppressants or in WBS from patients on immunosuppressive therapy (n=56). In addition, healthy controls (n=10) and patients without immunosuppression (n=10) were included. A comparison (n=15) with an established western blot method based on anti-phospho p70S6 kinase (Thr389) was made by splitting WBS.
Results: Everolimus decreased p-S6RP in vitro concentration dependently (0.00-27.4μg/L). This effect was also confirmed in vivo after a single dose of everolimus to healthy volunteers (n=3). However, spiking WBS with 500μg/L cyclosporine also decreased p-S6RP. The within-run coefficient of variation was <18% in transplant patients and <27% in healthy controls for both cell subsets. Sample stability for p-S6RP analysis was limited (<24h). p-S6RP was significantly decreased in CD3+CD8+ cells of patients treated with sirolimus (p=0.02) but not with everolimus. No significant correlation between the phosphoflow- and western blot method was noted.
Conclusion: The phosphoflow assay of p-S6RP performed well analytically, but sample stability, specificity, and method comparison results question its fitness for clinical purposes.
(Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)
Design and Methods: A kit was used for fixation and permeabilization of mitogen-stimulated cells, and p-S6RP was assessed separately in CD3+CD4+ and CD3+CD8+ cells by employing an anti-phospho-Ser235/236 antibody. Specificity, linearity, within-run precision and stability were investigated in either WBS spiked with everolimus and non-mTORi immunosuppressants or in WBS from patients on immunosuppressive therapy (n=56). In addition, healthy controls (n=10) and patients without immunosuppression (n=10) were included. A comparison (n=15) with an established western blot method based on anti-phospho p70S6 kinase (Thr389) was made by splitting WBS.
Results: Everolimus decreased p-S6RP in vitro concentration dependently (0.00-27.4μg/L). This effect was also confirmed in vivo after a single dose of everolimus to healthy volunteers (n=3). However, spiking WBS with 500μg/L cyclosporine also decreased p-S6RP. The within-run coefficient of variation was <18% in transplant patients and <27% in healthy controls for both cell subsets. Sample stability for p-S6RP analysis was limited (<24h). p-S6RP was significantly decreased in CD3+CD8+ cells of patients treated with sirolimus (p=0.02) but not with everolimus. No significant correlation between the phosphoflow- and western blot method was noted.
Conclusion: The phosphoflow assay of p-S6RP performed well analytically, but sample stability, specificity, and method comparison results question its fitness for clinical purposes.
(Copyright © 2016 The Canadian Society of Clinical Chemists. Published by Elsevier Inc. All rights reserved.)