Figure 3.

c-DDP induces Fas ligand expression through the activity of the JNK pathway. (a) Levels of expression of FasL after treatment with c-DDP. 293T cells were exposed to c-DDP at 10 μg/ml. At the indicated times cultured cells were collected, and FasL expression examined by immunoblotting using a polyclonal antiserum against FasL. (b) Regulation of the transcriptional activation of the FasL promoter by c-DDP and etoposide. 293T cells were transiently transfected with 1 μg of FasL-LUC reporter plasmid. Twenty-four hours after transfection, cells were treated with 2.5 μg/ml c-DDP or 5 mM Etoposide for different times. Cells were lysed and analyzed for luciferase activity. The fold increase in luciferase activity was calculated based on the values for untreated cells. These data are representative of three experiments. (c) Luciferase activity showing the effect of MEKK1 and MEKK1 (KR) expression on FasL promoter activation by stress stimuli. 293T cells were transiently cotransfected with 1 μg of FasL-Luc construct and 2 μg of MEKK1 or MEKK1 (KR). Twenty-four hours after transfection cells were treated with c-DDP. The cells were lysed 5 h later and analyzed for luciferase activity. Fold increase in luciferase activity was calculated based on the value for untreated control cells. (d and e) Contribution of AP1 and NFκB transcription factor on FasL promoter activation. 293T cells were transiently cotransfected with 1 μg of FasL-Luc (d, top panel) or HIV-LUC (d, bottom panel) construct and either 2 μg of pMEKK1, 2 μg of IKBα-SR, or 2 μg of CL100. Luciferase activity was analyzed as in c. Data shown in this figure represent the mean of a single experiment performed in triplicate ±SD and are representative of at least three experiments with similar results.