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doi: 10.1242/10.1242/jcs.00153

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Caspase-dependent initiation of apoptosis and necrosis by the Fas receptor in lymphoid cells: onset of necrosis is associated with delayed ceramide increase

¹ Instituto de Ciencias Biomédicas, Facultad de Medicina, Universidad de Chile, Santiago, Chile
² Institute of Biochemistry, University of Lausanne, Switzerland

View larger version (15K): [in a new window]   Fig. 1. Cell viability, caspase-3 activation, DNA fragmentation and surface exposure of phosphatidylserine in FasL-stimulated A20 B-lymphoma cells. (A) Cells were incubated for 30 minutes without (-) or with either 100 µM Ac-DEVD-cho (DEVD or D), 100 µM Ac-YVAD-cho (YVAD or Y) or 10 µM zVAD-fmk (zVAD or z). Then, cells were incubated for another 16 hours either without (white bars) or in the presence of 100 ng/ml of FasL (gray bars), and cell viability was determined by the MTS assay. (B) Caspase-3 activity was determined using the substrate Ac-DEVD-amc after incubation of cells (0-8 hours) with 10 (open circles), 32 (filled circles) or 100 (open squares) ng/ml FasL. (C) Cells were treated as described in (A), and after 4 hours of incubation with FasL, DNA fragmentation was analyzed by electrophoresis in agarose gels. (D) A20 cells were incubated (0 to 5 hours) with 100 ng/ml of FasL alone or 3 hours with FasL following pre-incubation with the inhibitor Ac-DEVD-cho (D) or zVAD-fmk (z), and then phosphatidylserine exposure on the cell surface was detected using Annexin-V-FITC. Only PI-negative cells, which represented approximately 90% of the total cell population, are shown. Data shown are the means with standard deviations from (A,B,D) or results representative of (C) three independent experiments.

View larger version (41K): [in a new window]   Fig. 5. Comparison of FasL-induced cell death in A20, Jurkat and Raji cells. (A) Cells were incubated either with increasing concentrations up to 400 ng/ml of FasL (A20, Jurkat cells) or up to 400 ng/ml of anti-Fas antibody (Raji cells) for 16 hours, stained with PI and cell death was assessed. (B) A20 and Jurkat cells were incubated with or without (NT) 100 ng/ml FasL after pre-treatment for 30 minutes with the indicated low concentrations (0-5 µM) of zVAD-fmk (zVAD). Cell death was quantified by flow cytometric analysis (see Fig. 2). Squares or empty bars indicate the percentage of apoptotic cells, and circles or gray bars indicate necrotic cells (A,B). Results averaged from three experiments are shown. Also, PS exposure on the cell surface of A20, Jurkat and Raji cells was detected by FACS using Annexin-V-FITC 5 hours following Fas activation (C). Subpopulations R1 and R2 indicated for A20 and Jurkat cells were obtained as described in Fig. 4. Results shown are representative of two independent experiments.

View larger version (20K): [in a new window]   Fig. 2. FasL induced both apoptotic and necrotic cell death in A20 B-lymphoma cells. Cells were incubated for 30 minutes with or without 100 µM Ac-DEVD-cho (DEVD) or 10 µM zVAD-fmk (zVAD). Then FasL was added to a final concentration of 100 ng/ml, and cells were incubated for another 16 hours. Subsequently, cells were stained with PI, and cell survival was determined by flow cytometric analysis. (A) In response to FasL, two populations of PI-positive, dead cells, indicated as M1 (hypodiploid population) and M2 (dead cells with intact DNA), were distinguishable. (B) Statistical analysis of results from experiments as indicated in A, showing the means with standard deviation of three independent experiments. As additional negative controls, results obtained with cells resistant to FasL (A20R) are also shown. (C) In parallel, cells analyzed as in A were permeabilized with methanol and stained with PI to quantify total DNA content. Data shown are representative of three independent experiments with similar outcomes.

View larger version (58K): [in a new window]   Fig. 3. Morphological analysis and DNA staining of A20 B-lymphoma cells treated with FasL. Untreated cells (A) or cells treated with 100 ng/ml FasL (B,C) for 16 hours were analyzed by electron microscopy. Necrotic FasL-treated cells (C) were characterized by disruption of the nuclei and abundance of vacuolar structures, whereas apoptotic cells observed in response to FasL were characterized by nuclear fragmentation, strong condensation of chromatin and membrane blebbing (B). The black bar shown for untreated cells (A) is equivalent to 5 µm. All images are shown at the same magnification. Alternatively, cells were left untreated (NT) or incubated with 100 ng/ml FasL for 16 hours, stained with PI and analyzed by confocal microscopy (D). As a comparison, non-treated cells are shown. Non-treated cells permeabilized with methanol (NT, Met-OH) and stained with PI (control, permeabilized) are shown as controls in Fig. 8A at the same magnification. Necrotic nuclei were PI positive, but retained a normal structural appearance (N); apoptotic nuclei were characterized by strong condensation of chromatin (A). Phase contrast and fluorescence images from the same optical section are shown. (E) Alternatively, hypodipliod and normodiploid cells were separated by FACS using PI fluorescence intensity as a parameter and analyzed subsequently by confocal microscopy (D). Images are shown at similar magnification. The bar in white is equivalent to 13 µm.

View larger version (38K): [in a new window]   Fig. 4. Caspase-3 and caspase-8 activity in FasL-stimulated A20 B-lymphoma cells. (A) Cells were incubated for 4 hours either without (NT) or in the presence of 100 ng/ml of FasL (FasL), and cell volumes were determined by FACS analysis by plotting forward scatter versus side scatter. After treatment with FasL, approximately 65% of cells showed a reduction in cell volume (Region 2, R2). This phenomenon was not observed in non-treated cells (Region 1, R1). (B) Caspase-3 and caspase-8 activity in situ were determined by flow cytometric analysis using the cell permeable substrates FAM-DEVD-fmk and FAM-LETD-fmk, respectively. Non-treated cells (dotted line), or cells treated with 100 ng/ml FasL from regions R1 (grey line) and R2 (black line) region, are shown. Alternatively, both cell populations R1 and R2 were separated by cell sorting. Then, either nuclear morphology was analyzed by confocal microscopy following permeabilization with methanol and PI staining (C) or DNA fragmentation was visualized following electrophoresis in agarose gels (D). In the latter experiments, cells treated with 100 ng/ml FasL (F) or left untreated (NT) served as controls. Results shown are representative of two independent experiments performed in duplicates.

View larger version (18K): [in a new window]   Fig. 6. Ceramide and diacylglycerol levels in FasL-stimulated A20 and A20R cells. After addition of 100 ng/ml FasL, A20 or A20R cells were incubated for the indicated time periods at 37°C. Lipids were extracted and analyzed using the diacylglycerol kinase assay and expressed as a percentage of basal levels. (A) Ceramide (open circles) and diacylglycerol (filled circles) levels 0-8 hours after FasL stimulation are shown in A20 cells. (B) Comparison in A20 and A20R cells of ceramide levels at the time points 3-8 hours following FasL addition. Results shown (means with standard deviation) were either from an individual experiment done in triplicate in which ceramide levels were already visibly elevated after 3 hours (A) or were averaged from two or more independent experiments done in triplicate for each time point.

View larger version (27K): [in a new window]   Fig. 7. Cell-permeable ceramides or treatment with bacterial SMase induced death by a caspase-3-independent mechanism. (A) A20 cells were incubated for 16 hours with 0-120 µM C6-ceramide (open circles) or dihydro-C6-ceramide (closed circles). Cell viability was determined by the MTS assay and expressed as percentage of cell viability observed for non-treated cells. (B) Caspase-3 activity was determined after treatment of A20 cells for 4 hours either with C6-ceramide at the concentrations indicated, with 80 µM dihydro-C6-ceramide (DH), with 100 ng/ml FasL (F), with 100 ng/ml FasL and 80 µM C6-ceramide (F/C6) or remained untreated. Values are shown as a percentage of the caspase-3 activity in the presence of 100 ng/ml FasL alone. (C) Alternatively, DNA fragmentation was analyzed by electrophoresis in agarose gels after treatment with FasL or different concentration of ceramides for 4 hours. (D) A representative experiment is shown where A20 cells were treated with 100 µM C6- (black) or dihydro-C6 (gray) ceramide for 16 hours and cell survival was determined by flow-cytometric analysis following PI staining. (E) A20, Jurkat and Raji cells were either not treated (NT) or treated with 100 µM C6-ceramides (C6) or 100 ng/ml FasL (F) for 16 hours. Then cells were harvested, stained with PI and cell survival was determined by flow cytometric analysis. (F) Cells were treated with bacterial SMase at the concentrations indicated for 16 hours and cell viability was determined by the MTS assay. (G) In parallel, cells either not treated (gray) or treated with 0.5 U/ml bacterial SMase (black) were stained with PI, and cell survival was determined by flow cytometric analysis. The results shown are either the means or standard deviations of three independent experiments (A,B,E,F) or are representative of at least three independent experiments (C,D,G).

View larger version (26K): [in a new window]   Fig. 8. Effect of cell permeable ceramides on FasL-induced cell death. (A) A20 or Raji cells (B) cells were incubated with 100 ng/ml FasL and C2-ceramide (100 µM) was added at time different time points after FasL. For comparison, cells were either not treated (NT or incubated with C2-ceramide (100 µM) or FasL (A20) or anti-Fas (Raji) (100 ng/ml) alone. For analysis, cells were harvested after 16 hours, stained with PI, and cell survival was determined by flow cytometric analysis (see Fig. 2). Means and standard deviations of data from three independent experiments are shown.

View larger version (68K): [in a new window]   Fig. 9. Morphological analysis and DNA staining of A20 B-lymphoma cells treated with cell-permeable ceramides. (A) Cells were incubated with 100 µM C2-ceramide for 16 hours, stained with PI and analyzed by confocal microscopy. As a comparison, non-treated cells were permeabilized with methanol (NT, Met-OH) and stained with PI (control, permeabilized). Phase contrast and fluorescence images from the same optical section are shown. The bar shown in white is equivalent to 13 µm. All images are shown at the same magnification. Alternatively, cells untreated (B) or treated with 100 µM C2-ceramide (C) for 16 hours were analyzed by electron microscopy. Necrotic C2-ceramide- (C) treated cells were both characterized by disruption of the nuclei and abundance of vacuolar structures. The black bar shown for untreated cells (B) is equivalent to 5 µm. The image in panel C is shown at the same magnification.

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