A spectrophotometric assay for routine measurement of mammalian target of rapamycin activity in cell lysates

• Published in: Analytical biochemistry 2010-08, Vol.403 (1-2), p.79-87
• Main Authors: Dekter, Hinke E., Romijn, Fred P.H.T.M., Temmink, Wouter P.M., van Pelt, Johannes, de Fijter, Johan W., Smit, Nico P.M.
• Format: Article
• Language: English
• Subjects: Animals; Cancer; CD3 Complex - immunology; Cell Line; Cells, Cultured; Drug monitoring; Drug Monitoring - methods; Enzyme assay; Enzyme-Linked Immunosorbent Assay - methods; Humans; Immunosuppressive agents; Immunosuppressive Agents - pharmacology; Intracellular Signaling Peptides and Proteins - antagonists & inhibitors; Intracellular Signaling Peptides and Proteins - blood; Intracellular Signaling Peptides and Proteins - metabolism; Kidney Transplantation; Leukocytes, Mononuclear - metabolism; Mammalian target of rapamycin; Phosphorylation; Protein kinase; Protein-Serine-Threonine Kinases - antagonists & inhibitors; Protein-Serine-Threonine Kinases - blood; Protein-Serine-Threonine Kinases - metabolism; Ribosomal Protein S6 Kinases, 70-kDa - metabolism; Sirolimus - pharmacology; T-Lymphocytes - immunology; T-Lymphocytes - metabolism; TOR Serine-Threonine Kinases; Transplantation; Transplantation, Homologous
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1016/j.ab.2010.04.022

The mammalian target of rapamycin (mTOR) is an important mediator in the PI3K/AKT signaling pathway. mTOR is the target of immunosuppressive drugs, such as rapamycin and everolimus, that are used in transplant patients but also for the treatment of various cancers. We have developed a method for mTOR activity measurement in cell lysates that measures the phosphorylation of p70 S6 kinase by an enzyme linked immunosorbent assay (ELISA) protocol. Using an optimized lysis composition, activity could be measured in the peripheral blood mononuclear cells (PBMCs) isolated from blood. For the PBMCs, intra- and interassay variations of 7 and 10%, respectively, were found using one lot number of the kit. With different lot numbers, the interassay variation increased up to 21%. Activity remained constant for PBMC pool samples on storage for a period of more than 7 months. Activity could also be measured in CD3+ T-cells isolated from blood. In vitro experiments revealed maximum mTOR inhibition of 30% in PBMCs and 44% in T-cells. The in vitro inhibition in PBMCs could also be demonstrated by Western blotting. The mTOR activity measurements may be used to show in vivo inhibition in renal allograft patients during everolimus treatment and to study mTOR activity in various (tumor) cell types.