Article Figures & Data
Figures
- FIGURE 1.
Effects of PKC signaling on cancer cell radioiodide uptake. (A) 125I uptake in T47D and PC12 cancer cells before and 48 h after infection with Ad.NIS. Cells were incubated with 74 kBq of 125I for 1 h and measured for uptake levels after rapid washing. Effects of PKC activation by 15-min stimulation with 100 ng of PMA per milliliter and PKC inhibition by 10 nM staurosporine or 6-h exposure to 100 ng of PMA per milliliter on 125I uptake in T47D/NIS (B) and PC12/NIS (C) cells. Data are mean ± SD of quadruplicate samples expressed as percentage uptake relative to that of control cells, obtained from single experiment representative of 2 separate experiments. †P < 0.005; ‡P < 0.0001, when compared with control cells. Staur = staurosporine.
- FIGURE 2.
Effect of EGF treatment on cancer cell radioiodide uptake and efflux. (A) Dose-dependent augmentation of 125I uptake in T47D/NIS and PC12/NIS cells by treatment with indicated concentrations of EGF for 6 h. (B) Time course of 10 nM EGF-stimulated 125I uptake in T47D/NIS and PC12/NIS cells. Data are mean ± SD of triplicate samples expressed as percentage uptake relative to untreated cells, obtained from single experiment representative of 3 separate experiments. (C) Effect of 6-h treatment with 10 nM EGF on radioiodide retention in T47D/NIS cells. After 1-h incubation with 125I, EGF-treated (EGF+) and -untreated cells (EGF−) were rapidly washed and incubated with fresh culture medium. Radioactivity remaining in cells over time was measured and expressed as mean ± SD of triplicate samples relative to that at time zero. *P < 0.001; †P < 0.0005; ‡P < 0.00005, when compared with untreated cells. NIS(−) = cells uninfected with Ad.NIS.
- FIGURE 3.
Effect of EGF treatment on ERK-1/2 activation. (A) T47D/NIS and PC12/NIS cell lysates underwent immunoprecipitation and immunoblotting with specific antibody against phosphorylated ERK-1/2 (p44 and p42 MAP kinase). Electrophoretic separation was done on 12% SDS-polyacrylamide gel. Group of cells was treated with 10 nM EGF for 30 min (EGF), and separate group of cells was cotreated with 25 μM of MEK inhibitor PD98059 (EGF+PD). (B) Quantitative protein band densities measured with calibrated densitometer. Data are mean ± SD of 3 samples per group expressed in arbitrary units. *P < 0.05; †P < 0.005, when compared with untreated cells.
- FIGURE 4.
Effect of MEK inhibitors on EGF-stimulated radioiodide uptake. Effects of graded concentrations of EGF or selective MEK1/2 inhibitor U0126 (25 μM) on 125I uptake in T47D/NIS (A) and PC12/NIS (B) cells are demonstrated. (C) Effect of MEK inhibitor PD98059 (25 μM) on125I uptake in T47D/NIS and PC12/NIS cells with or without 6-h treatment with 10 nM EGF is shown. All data are mean ± SD of triplicate samples expressed as percentage uptake relative to untreated cells, obtained from single experiment representative of 2 or 3 separate experiments.*P < 0.0005; †P < 0.0001, compared with cells treated with EGF without PD98059.
- FIGURE 5.
Immunoblots of membrane and total NIS protein. (A) Cell surface protein was prepared by biotinylation of T47D/NIS cells with 1 mg of sulfo-NHS-SS-biotin per milliliter and precipitation of surface-biotinylated protein with streptavidin-agarose beads. Cell surface and total NIS protein were separated on 10% SDS-polyacrylamide gel, electrotransferred, and immunoblotted with antihuman NIS antibody. (B) Quantitative NIS protein band densities were measured with calibrated densitometer. Data are mean ± SD of 4 samples per group expressed in arbitrary units. EGF = cells treated with 10 nM EGF for 6 h; EGF+PD = cells treated with EGF in presence of 25 μM PD98059.
- FIGURE 6.
(A) Immunofluorescent confocal microscopic localization of NIS protein. T47D/NIS cells grown on 8-well chamber slide were fixed in 2% paraformaldehyde and sequentially incubated with antihuman NIS antibody and Texas red–labeled secondary antibody. NIS localization was visually assessed under fluorescent confocal microscope (magnification, ×1200). EGF = cells treated with 10 nM EGF for 6 h; EGF+PD = cells treated with EGF in presence of 25 μM PD98059. (B) Effect of EGF on survival rate of 131I-treated cancer cells. T47D/NIS cells treated with 10 nM EGF for 6 h (EGF+) were compared with control cells (EGF−) for killing effect of exposure to 0, 0.74, or 1.48 MBq of 131I per milliliter for 7 h. Clonogenic assays were performed by seeding irradiated cells into 6-well plates at densities of 4,000 cells per well. Cells were fixed with methanol and stained with crystal violet, and number of colonies with 50 or more cells was counted after cells were cultured for 2 wk. Data are mean ± SD of 6 samples per group expressed as percentage of number of colonies for cells unexposed to 131I.
