American Society of Hematology

Figure 1.

$Figure 1. ALK+ ALCL cells are resistant to CD95/FAS-induced cell death as a result of c-FLIP overexpression. (A) Using trypan blue exclusion assay, cell viability did not change after treatment of 2 ALK+ ALCL cell lines, Karpas 299 and SU-DHL1, with increasing concentrations of the CH-11 antibody, an inducer of the extrinsic apoptotic pathway. These experiments were performed in triplicate, and the mean percentage of viable cells was calculated. Flow cytometry also showed no change in annexin V binding after treatment of Karpas 299 and SU-DHL1 cells with increasing concentrations of CH-11 (data not shown). By contrast, cell viability of the control CD30+ T-cell lymphoma cell line, Mac2a, decreased significantly after treatment of cells with increasing concentrations of the CH-11 antibody. Approximately 60% death of Mac2a cells was seen at a concentration of 200 ng/mL CH-11. Similar results were obtained for a control B-cell line, Mino (not shown). (B) Karpas 299 cells were transiently transfected with 5 μg or 10 μg c-FLIP siRNA, and whole-cell lysates were prepared at 48 hours after transfection. Immunoblots showed that endogenous c-FLIP expression was completely inhibited when 10 μg siRNA was used. (C) Transient transfection of Karpas 299 cells with 10 μg siRNA resulted in increased annexin V binding (from 21% to 39%) as shown by flow cytometry. Control transient transfections of Karpas 299 and SU-DHL1 cells with 10 μg or 20 μg siRNA (Ambion) of 2 housekeeping genes, GAPDH and 4EBP1, were also performed using the Nucleofector system (Amaxa Biosystems). No changes in cell viability and only minimal changes in the fraction of annexin V-positive cells were observed (not shown). These experiments were performed in triplicate. (D) Karpas 299 cells were initially transfected with 10 μg c-FLIP siRNA and, at 24 hours after transfection, the cells were treated with 200 ng/mL CH-11. As shown here, inhibition of c-FLIP expression significantly sensitized Karpas 299 cells to FAS-mediated apoptosis induced by CH-11. Cell viability was assessed by trypan blue exclusion studies. Bar graphs show results from experiments performed in triplicate using SU-DHL1 cells. Similar results were obtained using Karpas 299 cells. (E) Annexin V/propidium iodide (PI) staining assessed by flow cytometry showed a substantial increase of annexin V-positive (from 10% to 45%) and PI-positive SU-DHL1 cells. (F) Evidence of apoptotic morphology (nuclear condensation and fragmentation) in SU-DHL1 cells stained with 4′,6′-diamidino-2-phenylidole (DAPI) (top panel). Membranous and cytoplasmic expression of cleaved caspase-8 detected using an immunofluorescence method in SU-DHL1 cells that were treated with CH-11 and c-FLIP siRNA (bottom panel). Similar findings were seen using Karpas 299 cells. Immunofluorescence: × 400 original magnification. Images were obtained with a BX51 Olympus microscope and a DP12 Olympus camera (Olympus, Melville, NY). An Olympus universal semi-apochromat UPlan FI objective lens (40 ×/0.75 NA) was used. (G) Immunoblots showed that CH-11-induced apoptotic cell death is associated with cleavage of caspase-8 in Karpas 299 (and SU-DHL1) cells treated with c-FLIP siRNA. Error bars in panels A and D indicate standard error.$

ALK⁺ALCL cells are resistant to CD95/FAS-induced cell death as a result of c-FLIP overexpression. (A) Using trypan blue exclusion assay, cell viability did not change after treatment of 2 ALK⁺ ALCL cell lines, Karpas 299 and SU-DHL1, with increasing concentrations of the CH-11 antibody, an inducer of the extrinsic apoptotic pathway. These experiments were performed in triplicate, and the mean percentage of viable cells was calculated. Flow cytometry also showed no change in annexin V binding after treatment of Karpas 299 and SU-DHL1 cells with increasing concentrations of CH-11 (data not shown). By contrast, cell viability of the control CD30⁺ T-cell lymphoma cell line, Mac2a, decreased significantly after treatment of cells with increasing concentrations of the CH-11 antibody. Approximately 60% death of Mac2a cells was seen at a concentration of 200 ng/mL CH-11. Similar results were obtained for a control B-cell line, Mino (not shown). (B) Karpas 299 cells were transiently transfected with 5 μg or 10 μg c-FLIP siRNA, and whole-cell lysates were prepared at 48 hours after transfection. Immunoblots showed that endogenous c-FLIP expression was completely inhibited when 10 μg siRNA was used. (C) Transient transfection of Karpas 299 cells with 10 μg siRNA resulted in increased annexin V binding (from 21% to 39%) as shown by flow cytometry. Control transient transfections of Karpas 299 and SU-DHL1 cells with 10 μg or 20 μg siRNA (Ambion) of 2 housekeeping genes, GAPDH and 4EBP1, were also performed using the Nucleofector system (Amaxa Biosystems). No changes in cell viability and only minimal changes in the fraction of annexin V-positive cells were observed (not shown). These experiments were performed in triplicate. (D) Karpas 299 cells were initially transfected with 10 μg c-FLIP siRNA and, at 24 hours after transfection, the cells were treated with 200 ng/mL CH-11. As shown here, inhibition of c-FLIP expression significantly sensitized Karpas 299 cells to FAS-mediated apoptosis induced by CH-11. Cell viability was assessed by trypan blue exclusion studies. Bar graphs show results from experiments performed in triplicate using SU-DHL1 cells. Similar results were obtained using Karpas 299 cells. (E) Annexin V/propidium iodide (PI) staining assessed by flow cytometry showed a substantial increase of annexin V-positive (from 10% to 45%) and PI-positive SU-DHL1 cells. (F) Evidence of apoptotic morphology (nuclear condensation and fragmentation) in SU-DHL1 cells stained with 4′,6′-diamidino-2-phenylidole (DAPI) (top panel). Membranous and cytoplasmic expression of cleaved caspase-8 detected using an immunofluorescence method in SU-DHL1 cells that were treated with CH-11 and c-FLIP siRNA (bottom panel). Similar findings were seen using Karpas 299 cells. Immunofluorescence: × 400 original magnification. Images were obtained with a BX51 Olympus microscope and a DP12 Olympus camera (Olympus, Melville, NY). An Olympus universal semi-apochromat UPlan FI objective lens (40 ×/0.75 NA) was used. (G) Immunoblots showed that CH-11-induced apoptotic cell death is associated with cleavage of caspase-8 in Karpas 299 (and SU-DHL1) cells treated with c-FLIP siRNA. Error bars in panels A and D indicate standard error.

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