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...
Subarachnoid hemorrhage (SAH) often leads to a long-term narrowing of cerebra! artery called vasospasm. To understand the molecular mechanisms in vasospasm, signal transduction of tyrosine kinase pathway and phosphorylation of myosin light chain (MLC) and calponin (CaP) in the basilar artery were studied. Vasospasm was produced in the canine basilar artery by a two-hemorrhage method, and vasocontraction was induced by a local application of KCI or serotonin to the basilar artery after a transclival exposure. Intracellular substrates of tyrosine kinase pathway, including Shc, Rafl, and extracellular-regulated kinases in the basilar artery, were activated after SAH, and the activation of Shc suggests stimulation of signal transductions from tyrosine kinase receptors, G-coupled receptors, or both. The activation of tyrosine kinase pathway in vasospasm also was supported by dose-dependent dilation of the spastic basilar artery on days 0 and 7 by topical application of genistein, a tyrosine kinase inhibitor, and associated marked inhibition of tyrosine phosphorylation of intracellular substrates, including Shc. In addition, the generation of protein kinase M, catalytic fragment of protein kinase C(alpha) (PKC alpha), in vasospasm on days 0 and 7 was inhibited in response to genistein, indicating an inactivation of mu-calpain. It is suggested, therefore, that the reversal of vasospasm by genistein is closely associated with the restoration of intracellular Ca2+ levels. However, the increased activities of Raf1 and extracellular-regulated kinases in vasospasm were declined on day 7 compared with those on day 0 or 2, suggesting that the activation of tyrosine kinase pathway is more closely associated with the early stage of vasospasm than with the late stage of vasospasm. The analysis by pyrophosphate polyacrylamide gel electrophoresis (PPi-PAGE) demonstrated three MLC bands in vasospasm on days 2 and 7, as well as in KCI- and serotonin-induced vasocontraction. Since PPi-PAGE resolves smooth muscle MLC into three bands in the MLC kinase (MLCK)-mediated phosphorylation and into a single band in the PKC-mediated phosphorylation based on the phosphorylation state, the current results suggest that MLC in vasospasm is phosphorylated by MLCK but not by PKC. In basilar artery, CaP was significantly down-regulated, and in addition, significantly phosphorylated on serine and threonine residues only in vasospasm on days 2 and 7. Although the significance of CaP phosphorylations in vivo still is controversial, CaP down-regulation and phosphorylation may attenuate the inhibition of Mg(2+)-ATPase activity by CaP and induce a potential enhancement of smooth muscle contractility in delayed vasospasm. Since CaP is phosphorylated in vivo by PKC, activated PKC in vasospasm may phosphorylate CaP. Thus, SAH stimulates tyrosine kinase pathway to increase intracellular Ca2+ and activate PKC, and the former activates MLCK to phosphorylate MLC, whereas the latter phosphorylates CaP but not MLC.
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.
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