Marker Alias

FK506 binding protein 12-rapamycin associated protein 1 • RAFT1 • FK506 binding protein 12-rapamycin associated protein 2 • FRAP • FRAP1 • FRAP2 • RAPT1 • FKBP-rapamycin associated protein • FKBP12-rapamycin complex-associated protein 1 • rapamycin target protein • p70 S6 kinase • TOR • FLJ44809 • MTORC1 • MTORC2 • RPTOR • RAPTOR • KIAA1303 • mammalian target of rapamycin complex 1

Diagnostic/Research Products

Investigators at EMD Biosciences (San Diego, CA) developed an assay for the measurement of mTOR activity. A GST fusion of the classical mTOR substrate p70S6K is pr-bound to a microtiter plate coated with glutathione. Sample containing mTOR is added to the sample well in the presence of ATP and manganese containing assay buffer. Active mTOR phosphorylates p70S6K at Thr389. The phosphorylated substrate is detected with a phosphospecific anti-p70S6K(T389) antibody, followed by detection with HRP-antibody conjugate; relative activity is determined by reading absorbance. Using the assay, it was possible to measure kinase activity of a partially purified mTOR standard generated from an enriched rat brain fraction. The enriched mTOR standard contains both mTOR/Raptor and mTOR/Rictor complexes. The detected mTOR activity is inhibited by the general PI-3K inhibitor wortmannin as well as rapamycin/FKBP12 (IC50 of 8 nM), a specific inhibitor of the mTOR/Raptor complex. The assay was miniaturized to a well volume of 20 ul using a 384-well plate format and a fluorescent HRP substrate to improve sensitivity and dynamics. Additionally, mTOR activity was detected associated with various mTOR complexes immunoprecipitated from crude 293 cell lysates. Unlike the mTOR /Rictor complex, immunoprecipitation of active mTOR/Raptor requires use of a CHAPS containing lysis buffer to ensure capture of the intact complex. Phosphorylation of Ser473 on Akt was used to analyze the kinase activity associated with the mTOR/Rictor complex. This non-radioactive assay is useful for in vitro mTOR inhibitor screening and for assessing the regulation of mTOR in cell signaling (Harvey KJ, AACR07, Abs. 287).

Investigators at Louisiana State University Health Sciences Center (Shreveport, LA) examined if bioluminescent imaging, a sensitive and non-invasive method of detecting and quantifying minimal tumor cells, can be used to monitor the in vivo tumor response to mTOR inhibitor, CCI-779, in a preclinical nude mouse model of minimal residual disease in head and neck squamous cell carcinoma. A head and neck squamous cell carcinoma cell line, FaDu, was stably transfected with reporter gene luciferase. Five mice with transfected cells injected in dorsal flank were treated with CCI-779 and another 5 with control. Following luciferin injection, luciferase-expressing cells could be quantified using the Xenogen IVIS system at the region of interest. Immediately after surgery, no significant difference in the number of viable luciferase expressing cells implanted was detected between the control and treated mice. At three weeks post-surgery, CCI-779 prevented tumor formation and the average tumor volume in the treated mice was 35 + 13 mm3 compared to 408 + 40 mm3 in the control group. Bioluminescent imaging of longitudinal tumor growth showed significant differences in luciferase activity between the control and treated mice. Phosphorylation of eukaryotic initiation factor 4E-binding protein-1 (4E-BP1) in PBMC decreased after treatment, suggesting mTOR inhibition contributes to the antitumor effects. These temporal and spatial measurements of tumor growth to monitor therapeutic response using bioluminescent imaging corroborate with their previous measurements of tumor volume using calipers in a large sample size. This technology has great implications for accelerating the preclinical development of drugs targeting minimal residual disease (Nathan CO, etal, AACR06, Abs. 4470).

Current as of

May 06, 2010