Activation of the mitochondrial caspase cascade in the absence of protein synthesis does not require c-Jun N-terminal kinase
Section snippets
Cells and reagents
c-Jun (1–79)-GST, acetyl-Asp-Glu-Val-Asp-7-amino-4-(trifluoromethyl)coumarin and acetyl-Leu-Glu-His-Asp-7-amino-4-(trifluoromethyl)coumarin were obtained from Calbiochem (La Jolla, CA). SP600125 was from BIOMOL (Plymouth Meeting, PA). Other reagents were from Sigma Aldrich (St. Louis, MO). A549-S cells were established previously in our lab from the G4S subclone [32], and cultured in F-12K medium (GIBCO-BRL) supplemented with 10% fetal bovine serum, 100 U/ml penicillin, and 100 μg/ml streptomycin
Dose dependency and time course of cell death
The dose dependencies of the cell death-inducing activity of actinomycin D, anisomycin, emetine, and cycloheximide following 8 h of incubation were measured in order to determine suitable concentrations for subsequent studies (Fig. 1A). The approximate LD50 at 8 h for actinomycin D, anisomycin, and emetine were 0.8 μM (1 μg/ml), 38 μM (10 μg/ml), and 100 μM (55 μg/ml), respectively (Fig. 1A). These concentrations have previously been shown to inhibit protein synthesis [33], [34]. Based on these
Discussion
Persistent JNK activation has been suggested as a mechanism that drives cells into apoptosis in response to numerous stimuli [6], [9], [10]. Indeed, forced activation of the JNK pathway by overexpression through transfection is sufficient to induce apoptosis, [18], [20] and abrogation of the JNK signaling pathway has been shown to attenuate apoptosis in several circumstances [7], [10], [18], [19], [20], [21], [22]. Moreover, recent JNK1 and JNK2 double knockout studies clearly demonstrated that
Acknowledgements
This work was supported by Grants HL37556 and ES05511 from the National Institutes of Health. The authors thank Drs. Victor Darley-Usmar, Douglas Ruden, and Anup Ramachandran for valuable comments, and Dr. Marion Spell for FACS analysis.
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