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P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

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Related in: MedlinePlus

(a) Phospho-flow cytometry detected dose-dependent decrease of p70S6 kinase (p70S6K) phosphorylation in peripheral blood mononuclear cells (PBMCs) in vitro. MFI index: Mean fluorescence intensity index (*P < 0.05, †P < 0.01 vs. phorbol-12-myristate-13-acetate + ionomycin treated PBMCs); (b) A representative experiment about p70S6K was analyzed in parallel by Western blotting.
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Figure 4: (a) Phospho-flow cytometry detected dose-dependent decrease of p70S6 kinase (p70S6K) phosphorylation in peripheral blood mononuclear cells (PBMCs) in vitro. MFI index: Mean fluorescence intensity index (*P < 0.05, †P < 0.01 vs. phorbol-12-myristate-13-acetate + ionomycin treated PBMCs); (b) A representative experiment about p70S6K was analyzed in parallel by Western blotting.

Mentions: To confirm the validity of the phospho-flow assay in vitro experiments, the phosphorylation status of p70S6 in mitogen-stimulated PBMCs was incubated in presence or absence of rapamycin plus PMA and ionomycin for 40 h. Phospho-flow analysis revealed that addition of rapamycin reduced the p70S6K phosphorylation in PBMCs of healthy volunteers (n = 3) in a dose-dependent manner [Figure 4a] and this was confirmed in a representative Western blotting [Figure 4b].


P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.

Wang JY, Fan H - Chin. Med. J. (2015)

(a) Phospho-flow cytometry detected dose-dependent decrease of p70S6 kinase (p70S6K) phosphorylation in peripheral blood mononuclear cells (PBMCs) in vitro. MFI index: Mean fluorescence intensity index (*P < 0.05, †P < 0.01 vs. phorbol-12-myristate-13-acetate + ionomycin treated PBMCs); (b) A representative experiment about p70S6K was analyzed in parallel by Western blotting.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4834780&req=5

Figure 4: (a) Phospho-flow cytometry detected dose-dependent decrease of p70S6 kinase (p70S6K) phosphorylation in peripheral blood mononuclear cells (PBMCs) in vitro. MFI index: Mean fluorescence intensity index (*P < 0.05, †P < 0.01 vs. phorbol-12-myristate-13-acetate + ionomycin treated PBMCs); (b) A representative experiment about p70S6K was analyzed in parallel by Western blotting.
Mentions: To confirm the validity of the phospho-flow assay in vitro experiments, the phosphorylation status of p70S6 in mitogen-stimulated PBMCs was incubated in presence or absence of rapamycin plus PMA and ionomycin for 40 h. Phospho-flow analysis revealed that addition of rapamycin reduced the p70S6K phosphorylation in PBMCs of healthy volunteers (n = 3) in a dose-dependent manner [Figure 4a] and this was confirmed in a representative Western blotting [Figure 4b].

Bottom Line: The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001).The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01).There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

View Article: PubMed Central - PubMed

Affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.

ABSTRACT

Background: The phosphorylation of p70S6 kinase (p70S6K) represents an important target for sensitive detection on pharmacodynamic effects of sirolimus, but the methods of assessing p70S6K phosphorylation are still unclear. The aim of this study was to investigate p70S6K phosphorylation located down-stream of the mammalian target of rapamycin (mTOR) pathway in peripheral blood mononuclear cells (PBMCs) of liver transplant patients through different methods.

Methods: Seventy-five liver transplant recipients from Beijing Chaoyang Hospital of the Capital Medical University were analyzed in this study. Patients were divided into three groups, patient treated with sirolimus (n = 22), patient treated with tacrolimus (n = 30), patient treated with cyclosporine (n = 23). The p70S6K phosphorylation of PBMCs in patients and healthy control (HC, n = 12) were analyzed by phospho-flow cytometry and Western blotting. A correlation analysis of data from phospho-flow cytometry and Western blotting was performed. Intra-assay variability of p70S6K phosphorylation in HC and different patients were measured.

Results: Intra-assay variability of p70S6K phosphorylation in phospho-flow cytometry was from 4.1% to 8.4% and in Western blotting was from 8.2% to 18%. The p70S6K phosphorylation in patients receiving a sirolimus (19.5 ± 7.7) was significantly lower than in HC (50.1 ± 11.3, P < 0.001), tacrolimus (37.7 ± 15.7, P < 0.001) or cyclosporine treated patients (41.7 ± 11.7, P < 0.001). The p70S6K phosphorylation in HC (50.1 ± 11.3) was significantly higher than in tacrolimus (37.7 ± 15.7, P < 0.01) or cyclosporine-treated patients (41.7 ± 11.7, P < 0.01). There was correlation between data from phospho-flow cytometry and data from Western blotting (r = 0.88, P < 0.001).

Conclusions: The degree of mTOR inhibition by assessing p70S6K phosphorylation was established by phospho-flow cytometry and Western blotting. Assessment of p70S6K phosphorylation may play an adjunct role to on pharmacodynamically guide and individualize sirolimus based on immunosuppression.

Show MeSH
Related in: MedlinePlus