Figure 5.

The mTOR mutant reverts the anti-tumoral effect of rapamycin in HNSCC xenografts. A, The quantitative analysis of blood vessel density for each group was performed at the indicated days upon initiation of the rapamycin treatment as described above. Column, means blood vessel density in arbitrary unit; bars, SE. B, Tissue lysates extracted from HNSCC xenograft expressing mTOR-RR and control constructs (GFP) after treatment with vehicle control or rapamycin for 4 consecutive days were examined by Western blot analysis with antibodies against HIF-1α, S6, and its phosphorylated form, as indicated. C, Active caspase-3 immunofluorescence staining from GFP (control) and mTOR-RR group at the indicated days as described in A. The images from 8 different microscopic fields were taken and the fluorescence intensity analyzed by Metamorph 4.0 as detail described in Materials and Methods. Column, means fluorescence intensity in arbitrary unit; bars, SEM. D&E, HNSCC cell lines (HN12 and Cal27) expressing the mTOR-WT, mTOR-RR and GFP were injected subcutaneously into nude mice, and animals were treated with rapamycin or vehicle control. Tumor volumes were determined as described in Materials and Methods. Column, mean tumor volume of HN12 and CAL27 xenografts from the GFP, mTOR-WT and mTOR-RR groups at day 23. The mean tumor volume of the mTOR-RR groups at day 30 and 33 for HN12 and CAL27 HNSCC cells, respectively, when they achieve a volume similar to that of the their control xenografts, were also included; bars, SEM. The significance of the differences between each group was analyzed by ANOVA and Bonferroni's multiple comparison tests. Statistically significance at the indicated p value in each vehicle and rapamycin treated mice are shown, NS represents no significant difference between group.