title: P70S6 kinase phosphorylation: a new site to assess pharmacodynamy of sirolimus.
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.
authors.forename: Jun-Yu
authors.initials: JY
authors.lastname: Wang
authors.affiliation: Department of Hepatobiliary Surgery, Beijing Chaoyang Hospital, Capital Medical University, Beijing 100020, China.
authors.forename: Hua
authors.initials: H
authors.lastname: Fan
mesh_terms: D000293:Adolescent; D000328:Adult; D000368:Aged; D015153:Blotting, Western; D016572:Cyclosporine / Q000493:pharmacokinetics / Q000627:therapeutic use; D005260:Female; D005434:Flow Cytometry; D006801:Humans; D007166:Immunosuppressive Agents / Q000493:pharmacokinetics* / Q000627:therapeutic use; D007963:Leukocytes, Mononuclear / Q000201:enzymology; D016031:Liver Transplantation; D008297:Male; D008875:Middle Aged; D010766:Phosphorylation; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000378:metabolism*; D020123:Sirolimus / Q000493:pharmacokinetics* / Q000627:therapeutic use; D016559:Tacrolimus / Q000493:pharmacokinetics / Q000627:therapeutic use; D055815:Young Adult
chemical_list: D007166:Immunosuppressive Agents; D016572:Cyclosporine; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D020123:Sirolimus; D016559:Tacrolimus