Activation of the mitochondrial caspase cascade in the absence of protein synthesis does not require c-Jun N-terminal kinase

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Abstract

Prolonged activation of the c-Jun N-terminal kinase (JNK) has been suggested as a signal for apoptosis in response to a wide variety of stimuli. Using three cytocidal RNA or protein synthesis inhibitors (actinomycin D, anisomycin, and emetine), the potential role of JNK in activation of the mitochondrial apoptotic cascade was investigated in A549-S cells. Protein synthesis inhibition per se was not the cause of cell death as cycloheximide induced only growth arrest. All the cytocidal inhibitors induced cytochrome c release and caspases 9 activation within hours, but only anisomycin caused persistent JNK activation. Although, the JNK inhibitor, SP600125, inhibited JNK-dependent anisomycin-induced c-Jun phosphorylation, it was ineffective in preventing anisomycin-induced caspase activation and cell death. Thus, all three lethal macromolecule synthesis inhibitors can activate the mitochondrial apoptotic machinery independent of JNK activation, demonstrating that the mitochondrial apoptotic pathway can be activated independently of the JNK pathway in the absence of protein synthesis.

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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