Article Figures & Data
Figures
- FIGURE 1.
Differential kinetics of JNK activation in Jurkat cells by stress stimuli or by coligation of the TCR and CD28. A, Prolonged JNK activation by UV, gamma irradiation, anisomycin, and anti-Fas mAb. Aliquots of 5 × 106 Jurkat-tTA cells were either left untreated or were stimulated with 200 J/m2 of UV, 3300 rad of gamma irradiation, 1 μg/ml of anisomycin, or 100 ng/ml of anti-Fas mAb for the indicated periods of time. The cells were lysed and 200 μg of lysates were incubated with 10 μg of GST-c-Jun bound to glutathione beads. The kinase reaction was performed in the presence of 10 μCi [gamma-32P]ATP for 30 min. The proteins were separated by SDS-PAGE and exposed to x-ray film for 2 to 4 h. The variation in background activity is due to differences in the length of exposure. These experiments were repeated twice with similar results. B, Transient JNK activation by CD3 plus CD28 coligation. Jurkat-tTA cells were stimulated with 10 μg/ml of anti-CD3 (OKT3) plus 2 μg/ml of anti-CD28 (9.3) for the indicated time periods. JNK activity was determined as described above. Similar data were obtained in a repeat experiment.
- FIGURE 2.
shown in this experiment were reproduced three times. Fold increase in JNK activity was 6-fold for Tet(+) cells stimulated with PMA plus ionomycin (18,668 cpm), 8-fold for unstimulated DA-MEKK1 Tet(−) cells (24984), and 15-fold in Tet(−) cells stimulated with PMA plus ionomycin (47,670 cpm) (lanes 2, 3, and 4). C, Western blot showing that DA-MEKK1 expression does not activate the ERK cascade. DA-MEKK1 cells were grown in the presence (+) or absence (−) of 0.1 μg/ml tet for 24 h. The cells were lysed, and the cell lysates were analyzed by Western blotting with anti-ERK mAb. The hypomobility shift observed with anti-CD3 (OKT3) treatment (lanes 2 and 4) reflects ERK activation.
Inducible expression of DA-MEKK1 in Jurkat cells leads to the constitutive JNK activation. A, Luciferase assay to demonstrate tTA expression. Jurkat BMS2 cells were transfected with 10 μg of the pUHD15.1 plasmid, which encodes for the tTA. The cells were stably selected in 2 mg/ml of G418, and subclone D5 was transiently transfected with the reporter plasmid, pUHC13.3. The cells were grown in the presence (+) or absence (−) of 0.1 μg/ml of tet for 24 h, and assayed for luciferase activity. Fold increase was calculated using luciferase activity under tet(+) conditions as a baseline (1155 light units). B, In vitro kinase assay showing the constitutive activation of JNK by DA-MEKK1. Jurkat-tTA cells were cotransfected with 30 μg of pUHD10.3 into which DA-MEKK1 was subcloned and 5 μg of pTPH plasmid, which contains the hygromycin cassette. Following selection in 270 μg/ml of hygromycin, the cells were grown in the presence (tet(+)) or absence (tet(−)) of 0.1 μg /ml of tet for 24 h. Cells were either left untreated (lanes 1 and 3), or were stimulated for 10 min with 100 nM of PMA and 1 μg/ml of ionomycin at 37°C (lanes 2 and 4). Data
- FIGURE 3.
Expression of DA-MEKK1 induces cell death in parallel with JNK activation in Jurkat cells. A, Expression of DA-MEKK1 induces cell death of Jurkat cells in a time-dependent manner. DA-MEKK1 Jurkat cells were grown in the presence –♦– or absence –▪– of 0.1 μg/ml of tet for the indicated time period. Cell viability was measured by trypan blue exclusion. Duplicate counts were performed by two independent observers. Similar results were obtained in three separate experiments. B, In vitro JNK assay showing the time-dependent activation of JNK by DA-MEKK1. Duplicate aliquots of the cell populations shown above were lysed and assayed for JNK activity using the kinase assay described in Figure 1. No JNK activation was observed in DA-MEKK1 cells grown in the presence of tet (not shown)
- FIGURE 4.
Inducible expression of DA-MEKK1 leads to the apoptosis of Jurkat cells. A, DA-MEKK1-expressing cells exhibit morphologic characteristics of apoptosis. DA-MEKK1 cells were grown in the presence (tet(+)) or absence (tet(−)) of tet for the indicated times. Morphologic characteristics were examined under phase contrast microscopy 48 h after the removal of tet (original magnification ×200). B, FACS analysis showing that expression of DA-MEKK1 leads to intercalation of 7-AAD into cellular DNA. DA-MEKK1 Jurkat cells, grown in the presence or absence of tet, were stained with 1 μg/ml of 7-AAD 48 h after the withdrawal of tet, and analyzed by flow cytometry using the Lysis II program (Becton Dickinson). For comparison, Jurkat-tTA cells were treated with anti-Fas mAb (CH11) for 8 h. The percentages of apoptotic and live cells are included. These results were reproduced three times. C, DA-MEKK1 expression induces DNA laddering in Jurkat cells. Total DNA was extracted from DA-MEKK1 cells, separated by 2% agarose electrophoresis, and viewed by transillumination. Similar results were obtained in two additional experiments.
- FIGURE 5.
The induction of apoptosis by DA-MEKK1 is dependent on Fas and FasL. A, Immunostaining showing enhanced Fas and FasL expression in cells expressing DA-MEKK1. DA-MEKK1 cells, grown under off (tet(+)) or on (tet(−)) conditions for 36 h (top panel), were stained with anti-Fas (CH11) or anti-FasL (NOK1) mAb, followed by FITC-coupled anti-mouse Ig, and analyzed by flow cytometry using the CellQuest program (Becton Dickinson). For comparison, Jurkat-tTA cells were either left untreated or were stimulated with 100 nM of PMA plus 1 μg/ml of ionomycin for 12 h (bottom panel) and analyzed as above. B, RT-PCR showing the enhanced expression of FasL mRNA in cells expressing DA-MEKK1. DA-MEKK1 cells grown in the presence of tet were either left unstimulated or were treated with 100 nM of PMA plus 1 μg/ml of ionomycin for 6 h (lanes 1 and 2). Lanes 3 and 4 represent DA-MEKK1 cells from which tet was withdrawn for the indicated periods of time. RT-PCR analysis was performed using the primers for FasL and β-actin described in Materials and Methods. C, Inhibition of DA-MEKK1-mediated apoptosis by the Fas-Fc fusion protein. DA-MEKK1 cells were incubated in the presence or absence of 30 μg/ml of Fas-Fc and grown under (tet(+)) or (tet(−)) conditions for the indicated time periods. Cell viability was determined by trypan blue exclusion as described in Figure 3A.
- FIGURE 6.
Immunostaining showing the induction of FasL expression by stress stimuli. Jurkat-tTA cells were stimulated for 14 h with 200 J/m2 of UV, 3300 rad of gamma irradiation, 1 μg/ml of anisomycin in the presence of 30 μM of Z-VAD. This prevents induction of apoptosis by these stimuli (see Table III). DA-MEKK1-expressing cells were used as a positive control. Immunostaining and FACS analysis were performed as described in Figure 5A.
- FIGURE 7.
Regulation of the transcriptional activation of the FasL promoter by DA- and DN-MEKK1. A, Luciferase assay showing the regulation of the FasL promoter by DA-MEKK1. DA-MEKK1 cells, transiently transfected with 50 μg of FasL-Luc construct, were grown in the presence, tet(+), or absence, tet(−), of 0.1 μg/ml of tet for 24 h. Cells were left unstimulated or were treated with 10 μg/ml of anti-CD3, 100 nM of PMA plus 1 μg/ml of ionomycin for 8 h. The cells were lysed and 100 μg of cell lysates were analyzed for luciferase activity. The fold increase in luciferase activity was calculated based on the value for tet(+) cells, which amounted to 7584 relative light units. This data is representative of three experiments. B, Luciferase activity showing the effect of DN-MEKK1 on FasL promoter activation by stress stimuli. Jurkat-tTA cells were transiently cotransfected with 50 μg of FasL-Luc and 20 μg of DN-MEKK1. The cells were grown in the presence or absence of 0.1 μg/ml of tet for 24 h, and stimulated with 200 J/m2 of UV, 3300 rad of gamma irradiation, or 1 μg/ml of anisomycin in the presence of 30 mM of Z-VAD. The cells were lysed 8 h later and analyzed for luciferase activity. Fold increase in luciferase activity was calculated based on the value for unstimulated tet(+) cells, which amounted to 4865 relative light units.
- FIGURE 8.
Inhibition of apoptosis by Fas-Fc protein in cells stimulated with anisomycin but not UV or gamma irradiation. Jurkat-tTA cells were stimulated with 200 J/m2 of UV, 3300 rad of gamma irradiation, or 1 μg/ml of anisomycin in the presence or absence of 25 μM of Fas-Fc protein. DA-MEKK1 cells were grown in the absence of tet for 48 h. The cells were examined for apoptosis 8 h later. Cell viability (trypan blue exclusion) was determined by two independent observers. Data represent the mean of two experiments.
Tables
Percent Apoptotic Cells in the Culture UV 65.5 ± 2.3 Gamma irradiation 59.5 ± 3.1 Anisomycin 71.3 ± 2.6 anti-Fas 47.5 ± 4.2 -
a Jurkat tTA cells were stimulated with 200 J/m2 UV, 3300 rad gamma irradiation, 1 μg/ml anisomycin, or 100 ng/ml anti-Fas mAb for 8 h. Cell death was determined by trypan blue exclusion. Duplicate counts were performed by two independent observers.
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Hours of Stimulation Percent Apoptosis Following Stimulation with Anti-CD3 Anti-CD3 + anti-CD28 0 4.8 ± 1.8 4.3 ± 1.5 12 15.5 ± 2.3 3.4 ± 5.2 24 28.3 ± 4.6 3.4 ± 3.4 48 41.7 ± 2.5 6.1 ± 1.8 72 43.1 ± 1.8 7.9 ± 4.0 -
a Jurkat tTA cells were stimulated with 10 μg/ml anti-CD3 (OKT3) or a combination of 10 μg/ml anti-CD3 + 2 μg/ml anti-CD28 (9.3) for 24 h. Cell death was determined by trypan blue exclusion. Duplicate counts were performed by two independent observers.
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Stimulus Percent Apoptosis Following Stimulation Absence of Z-VAD Presence of Z-VAD Media 6.7 ± 1.3 5.9 ± 0.8 UV 68.9 ± 9.1 11.3 ± 1.8 Gamma irradiation 73.9 ± 5.5 12.7 ± 1.5 Anisomycin 86.1 ± 5.9 8.3 ± 0.6 anti-Fas 55.1 ± 0.9 7.6 ± 1.1 -
a Jurkat cells were stimulated in the presence or absence of 30 μM Z-VAD. The percentage of apoptotic cells was determined 16 h later.
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