The present research aimed at demonstrating the relationship between mTORC1 and mTORC2 inhibition induced Akt activation, and specially the biological importance of Akt class II HDAC inhibitor activation in mTOR targeted cancer therapy. METHODS AND materials For detailed information on Western blot analysis, cell lines, reagents, development inhibition assay, colony formation assay, cell cycle analysis, immunohistochemistry and figure analysis, please see text. Establishment of a Rapamycin resistant Cell Line The resistant A549 cell line was established by exposing the rapamycinsensitive A549 adult cells to gradually increased concentrations of rapamycin from the initial 1 nM to the remaining 20 uM over a 6 month period. A549 RR cells were routinely cultured in full medium containing 1 uM rapamycin. Immunoprecipitation mTOR complexes were immunoprecipitated with goat polyclonal mTOR antibody based on the same process described previously. Gene Knockdown by Small Interfering RNA Raptor and rictor siRNAs and lentiviral raptor, rictor and struggle shRNAs were produced from Qiagen and explained previously Endosymbiotic theory. For transfection and detail by detail sequences, please see Supplemental Text. Animal experiments were permitted by the Institutional Animal Care and Use Committee of Emory University. Four to 6 week old female athymic mice were obtained from Taconic and housed under pathogen free conditions in microisolator cages with laboratory chow and water ad libitum. A549 cells at 5 106 in serum free medium were injected s. D. into the flank region of nude mice. When cancers reached particular size ranges, the mice were randomized into four groups according Erlotinib clinical trial to tumefaction sizes and human body weights for the next treatments: car get a grip on, produced RAD001, LY294002 in DMSO, and the combination of RAD001 and LY294002. Tumefaction volumes were calculated with the formula V /6 and measured using caliper sizes once every two days. After a 14 day treatment, the rats were sacrificed with CO2. The tumors were then removed, weighed and frozen in liquid nitrogen or fixed with formalin. Certain portions of tumor cells from each tumor were homogenized in protein lysis buffer for preparation of whole cell protein lysates as described previously. American blotting effects were quantitated using Kodak Image Station 2000R. RESULTS Ramifications of Prolonged Treatment with mTOR Inhibitors on Akt Phosphorylation are Dose-dependent others and We previously showed that rapamycin induces a rapid and sustained increase in Akt phosphorylation in several kinds of cancer cells including breast, lung and prostate cancer cells. But, two recent studies show that prolonged treatment with mTOR inhibitors decrease Akt phosphorylation in certain cancer cell lines.