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. Author manuscript; available in PMC: 2012 Sep 16.

Published in final edited form as: Cancer Res. 2008 Feb 15;68(4):1144–1153. doi: 10.1158/0008-5472.CAN-07-1756

The lentiviral expression of the rapamycin-insensitive mTOR mutant (mTOR-RR) reverts the biochemical effects of rapamycin in HNSCC cells, and reveals that HNSCC cells are the primary targets of rapamycin in HNSCC xenografts. A, Cartoon shows the domain structure of mTOR. The rapamycin-insensitive mTOR mutant was obtained by mutating the amino acid at position Ser 2035 located in the FRB domain for Ile, which disrupts the binding of rapamycin-FKBP12 complex to wild type (wt) mTOR. B, The mTOR-RR was expressed in HNSCC cell lines by lentiviral gene delivery, and cells were left untreated (-) or exposed to rapamycin (RP)(+) for 30 min. The expression of mTOR and the levels of total and phosphorylated S6 were analyzed Western blotting. C, Cartoon depicting the experimental design. HNSCC cell lines expressing mTOR-RR and GFP or mTOR-WT as controls were injected into the flanks of nude mice. After tumor development, rapamycin treatment was initiated for 5 consecutive days. The tissues were collected at every day of treatment for further analysis. D, Top panel; Double IF staining of pS6 (in green) and CD31 (in red) were performed after two days of rapamycin treatment. Note the reduction of pS6 in the tissues from the control group expressing GFP (a), but not after expressing the mTOR-RR (b). The architecture of cancer and endothelial cells within the tumor mass was documented by the fluorophore labeling by E-cadherin (in green) and CD31 (in red), respectively, as shown in panels c and d. In parallel, the apoptotic response to rapamycin treatment detected by the IF staining of cleaved caspase-3 (in red) was found to overlap with the E-cadherin staining (in green) of tumor cells (e) in the control group but not in xenografts of mTOR-RR expressing cells (f).

The lentiviral expression of the rapamycin-insensitive mTOR mutant (mTOR-RR) reverts the biochemical effects of rapamycin in HNSCC cells, and reveals that HNSCC cells are the primary targets of rapamycin in HNSCC xenografts. A, Cartoon shows the domain structure of mTOR. The rapamycin-insensitive mTOR mutant was obtained by mutating the amino acid at position Ser 2035 located in the FRB domain for Ile, which disrupts the binding of rapamycin-FKBP12 complex to wild type (wt) mTOR. B, The mTOR-RR was expressed in HNSCC cell lines by lentiviral gene delivery, and cells were left untreated (-) or exposed to rapamycin (RP)(+) for 30 min. The expression of mTOR and the levels of total and phosphorylated S6 were analyzed Western blotting. C, Cartoon depicting the experimental design. HNSCC cell lines expressing mTOR-RR and GFP or mTOR-WT as controls were injected into the flanks of nude mice. After tumor development, rapamycin treatment was initiated for 5 consecutive days. The tissues were collected at every day of treatment for further analysis. D, Top panel; Double IF staining of pS6 (in green) and CD31 (in red) were performed after two days of rapamycin treatment. Note the reduction of pS6 in the tissues from the control group expressing GFP (a), but not after expressing the mTOR-RR (b). The architecture of cancer and endothelial cells within the tumor mass was documented by the fluorophore labeling by E-cadherin (in green) and CD31 (in red), respectively, as shown in panels c and d. In parallel, the apoptotic response to rapamycin treatment detected by the IF staining of cleaved caspase-3 (in red) was found to overlap with the E-cadherin staining (in green) of tumor cells (e) in the control group but not in xenografts of mTOR-RR expressing cells (f).