PMC

Proposed model for signal transduction pathways upon death receptor ligation.

Expression of DR5 and TRAIL by human astroglioma cells. (A) FACS analysis of DR5 expression on CRT-MG and U87-MG cells was performed as described in Materials and Methods. Thin lines indicate negative controls stained with secondary antibody conjugated to PE after incubation with an isotype-matched (IgG1) control antibody. (B) Cell lysates were prepared from CRT-MG and U87-MG cells, and 100 μg of protein was electrophoresed in 10% sodium dodecyl sulfate gels. Western blot analysis for TRAIL protein expression was done as described in Materials and Methods. The data are representative of three independent experiments.

Susceptibility to DR5-induced cell death and activation of caspase 3. (A) U87-MG (▪) and CRT-MG (▴) cells were incubated with various concentrations of anti-DR5 antibody TRA-8 (0 to 1,000 ng/ml) or hrTRAIL (0 to 200 ng/ml) for 24 h, and then cell death was measured by staining with Annexin V-FITC and PI. Data are the mean and SD for duplicate samples from one experiment and are representative of three independent experiments. (B) Cells were incubated with TRA-8 (100 ng/ml) in complete medium for various times (0 to 24 h), and caspase 3 activity was measured as indicated in Materials and Methods. The relative fluorescence of each sample was normalized to the amount of total protein. Data are the mean and SD for quadruplicate samples from one experiment and are representative of two independent experiments.

Induction of IL-8 expression upon DR5 ligation. (A) CRT-MG cells were treated with TRA-8 (100 ng/ml) or hrTRAIL (50 ng/ml) for various times (0 to 24 h), and IL-8 mRNA expression was measured by an RPA. Fold induction of IL-8 mRNA expression was normalized to the level of glyceraldehyde-3-phosphate dehydrogenase mRNA expression. The data are representative of three independent experiments. Cont, control. (B) CRT-MG cells were transiently transfected with an IL-8 promoter-luciferase construct and the pCMV-β-galactosidase construct as indicated in Materials and Methods. The cells were incubated with hrTRAIL (0 to 100 ng/ml) for 24 h, harvested, and then examined for luciferase and β-galactosidase activities. The luciferase activity of each sample was normalized to the β-galactosidase activity, and fold induction values were calculated as the ratio of the normalized luciferase activity of each sample to that of the control. RLA, relative luciferase activity. Data are the mean and SD for duplicate samples from one experiment and are representative of three experiments. (C) CRT-MG and U87-MG cells were treated with hrTRAIL (0 to 100 ng/ml) for 36 h, and supernatants were examined for IL-8 protein expression by an ELISA. IL-8 expression was normalized to the amount of total protein. Data are the mean and SD for duplicate samples from one experiment and are representative of three independent experiments. (D) CRT-MG cells stably transfected with construct IL-8p-d2EGFP were treated with hrTRAIL (50 ng/ml) for 36 h, stained with Annexin-V-PE, and then analyzed by FACS. The percentage of cells in each quadrant is indicated. GFP, green fluorescent protein. The data are representative of three independent experiments.

Effect of the nonspecific caspase inhibitor Boc-D-Fmk on apoptosis and IL-8 induction upon DR5 ligation. (A) CRT-MG cells were incubated in the absence or presence of the cell-permeable nonspecific caspase inhibitor Boc-D-Fmk (0 to 50 μM) for 3 h prior to a 24-h treatment with hrTRAIL (50 ng/ml), and cell death was evaluated by staining with Annexin-V and PI as indicated in Materials and Methods. Data are the mean and SD for duplicate samples from one experiment and are representative of three independent experiments. (B) CRT-MG cells were pretreated in the absence (second lane) or presence (third lane) of Boc-D-Fmk (50 μM) for 3 h, washed twice, and then treated with hrTRAIL (50 ng/ml) for an additional 24 h. IL-8 mRNA expression was measured by an RPA; fold induction of IL-8 is indicated. Data are representative of three experiments. GAPDH, glyceraldehyde-3-phosphate dehydrogenase. (C) CRT-MG cells were stably transfected with construct IL-8p-d2EGFP as indicated in Materials and Methods. The stable reporter cells were incubated in the absence or presence of Boc-D-Fmk (0 to 50 μM) for 3 h and treated with hrTRAIL (100 ng/ml) for an additional 24 h, and then EGFP expression was examined by FACS analysis. Data are the mean and SD for duplicate samples from three independent experiments. The asterisk indicates a P value of ≤0.05. (D) Cells were incubated in the absence or presence of Boc-D-Fmk (50 μM) for 3 h and washed twice prior to a 36-h treatment with hrTRAIL (50 ng/ml), and IL-8 protein expression was measured by an ELISA. Data are the mean and SD for quadruplicate samples from one experiment. Values significantly different from the values for the hrTRAIL-treated sample without Boc-D-Fmk pretreatment are indicated by an asterisk (P ≤ 0.05). The data are representative of two independent experiments.

Differential effects of caspase inhibitors on TRAIL-mediated apoptosis and IL-8 induction. (A) CRT-MG cells were incubated in the absence or presence of various caspase inhibitors (10 μM) for 3 h and treated with hrTRAIL (0 to 100 ng/ml) for 24 h, and then cell death was measured by staining with Annexin V-FITC and PI. The data are representative of three independent experiments. (B) CRT-MG cells were incubated with medium or hrTRAIL (100 ng/ml) for 24 h, and 40 μg of lysates was examined by Western blot analysis with antibodies against caspases (Casp) 1, 3, and 8 and PARP. The arrows indicate the precursors of caspases 1 (45 kDa), 3 (32 kDa), and 8 (55 kDa) and PARP (116 kDa). The arrowheads indicate proteolytic products of caspases 1 (20 and 30 kDa), 3 (17 and 22 kDa), and 8 (23 and 40 kDa) and PARP (85 kDa). The data are representative of three independent experiments. (C) CRT-MG cells were incubated in the absence or presence of various caspase inhibitors (50 μM) for 3 h and treated with hrTRAIL (100 ng/ml) for 24 h, and then 40 μg of lysates was examined by caspase 3 Western blot analysis. The upper panel indicates short-term exposure of the blot, and the lower panel shows prolonged exposure of the same blot. The arrows indicate unprocessed procaspase 3 (32 kDa), and the arrowheads indicate processed active caspase 3 products (17 kDa). The data are representative of three independent experiments. (D) CRT-MG cells stably transfected with the IL-8 promoter-luciferase construct were incubated in the absence or presence of various caspase inhibitors (10 μM) for 3 h and treated with hrTRAIL (100 ng/ml) for 24 h, and then lysates were examined for luciferase activity. The luciferase activity of each sample was normalized to the amount of total protein, and fold induction values were calculated as the ratio of the luciferase activity of each sample normalized to that of the control. Data are the mean and SD for duplicate samples from one experiment. Values significantly different from values for the hrTRAIL-treated sample without inhibitor treatment are indicated by asterisks (P ≤ 0.05). The data are representative of three experiments. (E) CRT-MG cells were incubated in the absence or presence of various caspase inhibitors (10 μM) for 3 h and treated withhrTRAIL (100 ng/ml) for 1 h, and then 5 μg of nuclear extracts was examined for AP-1 and NF-κB binding by an EMSA as indicated in Materials and Methods. Fold induction values were calculated as the ratio of the value for each sample to that for the control. The data are representative of three independent experiments. (F) An CRT-MG cell clone stably transfected with construct IL-8p-d2EGFP was incubated in the absence or presence of various caspase inhibitors (25 μM) for 3 h, treated with hrTRAIL (100 ng/ml) for 24 h, and analyzed by FACS after staining with Annexin V-PE. The diagrams on the left indicate the results of FACS: EGFP expression (IL-8 promoter driven) versus Annexin V-PE (apoptosis). The histograms in the middle indicate the EGFP expression of living cells (after selection of Annexin V-PE-negative cells). The MFI is indicated. The histograms on the right show Annexin V-PE staining. The data are representative of three experiments.