title: Development of a sensitive phospho-p70 S6 kinase ELISA to quantify mTOR proliferation signal inhibition.
abstract: BACKGROUND
  Drug blood levels can only serve as a surrogate because of the lack of information on the drug's direct pharmacological effects in the individual patient. Measurement of the mammalian target of rapamycin (mTOR) activity dependent on the phosphorylation status of p70 S6 kinase (p70 S6K) offers a practical way for monitoring pharmacodynamic drug activity, with the potential to better assess the state of immunosuppression in individual patients.
  
  
  METHODS
  Here, we established a novel in vitro model system by treating Jurkat cells and peripheral blood mononuclear cells with different concentrations of sirolimus after stimulation with phorbol 12-myristate 13-acetate.
  
  
  RESULTS
  A dose-dependent reduction of the p70 S6K phosphorylation status was demonstrated by Western blot and a newly established enzyme-linked immunosorbent assay (ELISA). Relative phospho-p70 S6K values from ELISA and relative densities from Western blot analysis in peripheral blood mononuclear cells revealed a strong correlation (Spearman correlation coefficient r s = 0.7, P = 0.01). Finally, parallel assays confirmed a sirolimus dose-dependent reduction of cytokine production and cell proliferation in the in vitro model.
  
  
  CONCLUSIONS
  Pharmacodynamic monitoring of mTOR inhibition with a p70 S6K ELISA could guide mTOR inhibitor immunosuppression therapy toward a more individualized therapy. The usage of this technique now has to be evaluated in a clinical series of patients.
authors.affiliation: Medical Department I, Nephrology Section, Ulm University, Ulm, Germany. bertram.hartmann@kfh-gesundheitszentrum.de
authors.forename: Bertram
authors.initials: B
authors.lastname: Hartmann
authors.forename: Xuemei
authors.initials: X
authors.lastname: He
authors.forename: Frieder
authors.initials: F
authors.lastname: Keller
authors.forename: Michael
authors.initials: M
authors.lastname: Fischereder
authors.forename: Markus
authors.initials: M
authors.lastname: Guba
authors.forename: Holger
authors.initials: H
authors.lastname: Schmid
mesh_terms: D049109:Cell Proliferation / Q000187:drug effects; D004305:Dose-Response Relationship, Drug; D004797:Enzyme-Linked Immunosorbent Assay / Q000379:methods / Q000592:standards; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology; D019169:Jurkat Cells; D007963:Leukocytes, Mononuclear / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism; D038762:Ribosomal Protein S6 Kinases, 70-kDa / Q000037:antagonists & inhibitors / Q000378:metabolism*; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology*; D020123:Sirolimus / Q000494:pharmacology; D058570:TOR Serine-Threonine Kinases / Q000037:antagonists & inhibitors* / Q000378:metabolism
chemical_list: D007166:Immunosuppressive Agents; C546842:MTOR protein, human; D038762:Ribosomal Protein S6 Kinases, 70-kDa; D058570:TOR Serine-Threonine Kinases; D020123:Sirolimus