Abstract
Apoptosis was induced in human glioma cell lines by exposure to 100 nM calphostin C, a specific inhibitor of protein kinase C. Calphostin C-induced apoptosis was associated with synchronous down-regulation of Bcl-2 and Bcl-xL as well as activation of caspase-3 but not caspase-1. The exposure to calphostin C led to activation of stress-activated protein kinase/c-Jun NH2-terminal kinase (SAPK/JNK) and p38 kinase and concurrent inhibition of extracellular signal-regulated kinase (ERK). Upstream of ERK, Shc was shown to be activated, but its downstream Raf1 and ERK were inhibited. The pretreatment with acetyl-Tyr-Val-Ala-Asp-aldehyde, a relatively selective inhibitor of caspase-3, or benzyloxycarbonyl-Val-Ala-Asp-fluoromethylketone (z-VAD.fmk), a broad spectrum caspase inhibitor, similarly inhibited calphostin C-induced activation of SAPK/JNK and p38 kinase as well as apoptotic nuclear damages (chromatin condensation and DNA fragmentation) and cell shrinkage, suggesting that caspase-3 functions upstream of SAPK/JNK and p38 kinase, but did not block calphostin C-induced surface blebbing and cell death. On the other hand, the inhibition of SAPK/JNK by transfection of dominant negative SAPK/JNK and that of p38 kinase by SB203580 induced similar effects on the calphostin C-induced apoptotic phenotypes and cell death as did z-VAD.fmk and acetyl-Tyr-Val-Ala-Asp-aldehyde, but the calphostin C-induced PARP cleavage was not changed, suggesting that SAPK/JNK and p38 kinase are involved in the DNA fragmentation pathway downstream of caspase-3. The present findings suggest, therefore, that the activation of SAPK/JNK and p38 kinase is dispensable for calphostin C-mediated and z-VAD.fmk-resistant cell death.
Highlights
Strongly stimulated by activation of protein-tyrosine kinase receptors [1] and activated by both Ras-dependent [2,3,4] and Ras-independent signalings [5] in response to activation of G protein-coupled receptors, and common intermediates in intracellular signaling cascades are involved in diverse cellular functions including growth and differentiation [6]
The inhibition of extracellular signal-regulated kinase (ERK) activity has been reported to correlate with the activation of SAPK/JNK and p38 kinase as well as with the induction of apoptosis in nerve growth factor-deprived PC12 pheochromocytoma cells [22], Fas-induced Jurkat cells [15, 24], and UVirradiated mouse fibroblasts [25], and the balance between the activity of the stress kinases to that of ERK has been proposed to determine cell fate [26], because the Raf1-ERK signaling pathway plays a pivotal role in suppressing apoptotic death [27, 28]
Calphostin C-induced Cellular Responses Are Partly Inhibited, but Cell Death Is Not Inhibited by z-VAD.fmk, Ac-DEVD.cho, Transfection of Dominant Negative SAPK/JNK, or SB203580 —To dissect calphostin C-induced apoptotic signaling pathways, we investigated the effects of three caspase inhibitors (100 M z-VAD.fmk, 100 M Ac-DEVD.cho, and 100 M Ac-YVAD.cho) on calphostin C-induced cellular responses by adding to the culture medium 30 min prior to the treatment with calphostin C. z-VAD.fmk and Ac-DEVD.cho demonstrated similar effects on calphostin C-mediated cellular responses; they inhibited cell shrinkage, but surface blebs still remained unchanged in number in both U-87MG and T98G cells as revealed by phase contrast images (Fig. 1, e, eЈ, g, and gЈ)
Summary
Strongly stimulated by activation of protein-tyrosine kinase receptors [1] and activated by both Ras-dependent [2,3,4] and Ras-independent signalings [5] in response to activation of G protein-coupled receptors, and common intermediates in intracellular signaling cascades are involved in diverse cellular functions including growth and differentiation [6]. The inhibition of ERK activity has been reported to correlate with the activation of SAPK/JNK and p38 kinase as well as with the induction of apoptosis in nerve growth factor-deprived PC12 pheochromocytoma cells [22], Fas-induced Jurkat cells [15, 24], and UVirradiated mouse fibroblasts [25], and the balance between the activity of the stress kinases to that of ERK has been proposed to determine cell fate [26], because the Raf1-ERK signaling pathway plays a pivotal role in suppressing apoptotic death [27, 28]. To characterize the molecular mechanism that regulates calphostin C-induced apoptosis of gliomas, the contributions of mitogen-activated protein kinase family members to cell death caused by calphostin C-induced down-regulation of Bcl-2 are explored using various inhibitors of caspases, SAPK/JNK, and p38 kinase in this work
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