The proteasome inhibitors lactacystin and AcLLNal induced p53-independent apoptosis in two human glioma cell lines, and the apoptosis was accompanied by up-regulation of immunoreactive wild-type p53, p21 fI , Mdm2, and p27 uipI . Pretreatment with cycloheximide decreased the induction of cell death independently of p53 protein status, suggesting that the upregulation of short-lived proteins is associated with proteasome inhibitor-induced apoptosis. Caspase-3-like proteases were activated in the proteasome inhibitor-mediated apoptosis, and the induction of cell death was inhibited more effectively in the presence of z-VAD.fmk than in the presence of Ac-DEVD.fmk, suggesting that caspases other than caspase-3 are involved. Nonetheless, there were no significant alterations in levels of immunoreactive Bcl-2, Bcl-x v , Bax, Bad, and Bak, nor any evidence of cytochrome c release into cytosol and dissipation of v v8 8 m . Thus, the proteasome inhibitor-induced apoptosis is mediated by a mitochondria-independent mechanism, and the once activated caspase-3 does not cause the cytochrome c release and the v v8 8 m disruption. z 1999 Federation of European Biochemical Societies.

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-x L 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 NH 2 -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 Cinduced 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.It has been expected that signal transduction pathways involving specific protein kinases are involved in mediating apoptosis. Extracellular signal-regulated kinase (ERK) 1 is most strongly stimulated by activation of protein-tyrosine kinase receptors (1) and also activated by both Ras-dependent (2-4) 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). Activation of PKC by 12-O-tetradecanoylphorbol-13-acetate results in the activation of Raf1 (7) and ERK (8 -11) within minutes, suggesting the involvement of PKC in the signaling pathway leading to ERK activation. In addition, Ras functions as an essential transducer of various physiological signals leading to cell growth and proliferation in a PKC-dependent or PKC-independent manner (12), and activated Ras also renders cells susceptible to apoptosis after depression of PKC activity (13,14).SAPK/JNK and p38 kinase have been proposed to mediate apoptosis, but a number of reports have challenged the notion that the activation of SAPK/JNK and/or p38 kinase is sufficient to induce apoptosis (15-21), and the integration and balance of SAPK/JNK and p38 pathways probably contribute to commitment to a...

Calphostin C acts at the regulatory domain as a highly selective inhibitor of protein kinase C (PKC), and staurosporine acts at the catalytic domain as a nonspecific PKC inhibitor. The authors investigated the capacity of calphostin C and staurosporine to promote apoptotic fragmentation of DNA in four human glioma cell lines. The exposure of glioma cell lines to 100 nM calphostin C for 2 to 8 hours induced a decrease in particulate PKC activities and exposure for 16 to 24 hours produced a concentration-dependent increase in internucleosomal DNA cleavage on agarose gel electrophoresis. In addition, the human glioma cells showed the classic morphological features of apoptosis: cell shrinkage, nuclear condensation, and the formation of apoptotic bodies. A 24-hour exposure to staurosporine failed to induce internucleosomal DNA fragmentation at concentrations generally used to achieve maximum inhibition of enzyme activity (50 nM) but promoted fragmentation at considerably higher concentration (more than 200 nM). Deoxyribonucleic acid fragments obtained from cells exposed to 100 nM calphostin C for 16 to 24 hours possessed predominantly 5'-phosphate termini, consistent with the action of a Ca++/Mg(++)-dependent endonuclease. Northern and Western blot analyses revealed that the exposure to 100 nM calphostin C for 4 hours failed to alter bcl-2 transcript and protein, but exposure for more than 8 hours decreased the amount of bcl-2 transcript and protein. Together, these observations suggest that calphostin C is capable of inducing apoptotic DNA fragmentation and cell death in a highly concentration dependent manner in human glioma cells and that the apoptosis is closely associated with the decrease in transcription and translation of bcl-2.

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Calphostin C-mediated apoptosis in glioma cells was reported previously to be associated with down-regulation of Bcl-2 and Bcl-x L . In this study, we report that 100 nM calphostin C also induces translocation and integration of monomeric Bax into mitochondrial membrane, followed by cytochrome c release into cytosol and subsequent decrease of mitochondrial inner membrane potential (DCm) before activation of caspase-3. The integration of monomeric Bax was associated with acquirement of alkali-resistance. The translocated monomeric Bax was partly homodimerized after cytochrome c release and decrease of DCm. The translocation and homodimerization of Bax, cytochrome c release, and decrease of DCm were not blocked by 100 mM z-VAD.fmk, a pan-caspase inhibitor, but the homodimerization of Bax and decrease of DCm were inhibited by 10 mM oligomycin, a mitochondrial F 0 F 1 -ATPase inhibitor. Therefore, it would be assumed that mitochondrial release of cytochrome c results from translocation and integration of Bax and is independent of permeability transition of mitochondria and caspase activation, representing a critical step in calphostin C-induced cell death. Cell Death and Differentiation (2000) 7, 511 ± 520.