Hideyasu Ikemoto

Proteasome inhibitors induce mitochondria-independent apoptosis in human glioma cells

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, p21Waf1, Mdm2, and p27Kip1. Pretreatment with cycloheximide decreased the induction of cell death independently of p53 protein status, suggesting that the up‐regulation 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‐xL, Bax, Bad, and Bak, nor any evidence of cytochrome c release into cytosol and dissipation of ΔΨ 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 ΔΨ m disruption.

Activation of Stress-activated Protein Kinase/c-Jun NH2-terminal Kinase and p38 Kinase in Calphostin C-induced Apoptosis Requires Caspase-3-like Proteases but Is Dispensable for Cell Death

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.

Calphostin C-mediated translocation and integration of Bax into mitochondria induces cytochrome c release before mitochondrial dysfunction

Calphostin C-mediated apoptosis in glioma cells was reported previously to be associated with down-regulation of Bcl-2 and Bcl-xL. 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 (ΔΨm) 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 ΔΨm. The translocation and homodimerization of Bax, cytochrome c release, and decrease of ΔΨm were not blocked by 100 μM z-VAD.fmk, a pan-caspase inhibitor, but the homodimerization of Bax and decrease of ΔΨm were inhibited by 10 μM oligomycin, a mitochondrial F0F1-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

Proteasome inhibitors induce Fas-mediated apoptosis by c-Myc accumulation and subsequent induction of FasL message in human glioma cells

Proteasome inhibitors were shown previously to induce mitochondria‐independent and caspase‐3‐dependent apoptosis in human glioma cell lines by unknown mechanisms. Here, we showed that treatment with proteasome inhibitors, lactacystin or acetyl‐leucinyl‐leucinyl‐norleucinal, led to elevation of the steady‐state c‐Myc protein but not c‐myc mRNA, suggesting the accumulation of c‐Myc protein by proteasome inhibitors. In addition, the marked association of c‐Myc protein with ubiquitin by treatment with proteasome inhibitors indicated the involvement of proteasome in c‐Myc proteolysis and the stabilization of c‐Myc protein by proteasome inhibitors in vivo. The expression of Fas (also termed CD95 or APO‐1) mRNA, if analyzed by reverse transcriptase polymerase chain reaction assay, was found to occur constitutively, and increased slightly by the treatment with proteasome inhibitors. In contrast, the expression of Fas ligand (FasL) mRNA was markedly induced temporarily before the activation of caspase‐3 by the treatment. Agonistic anti‐Fas antibody (CH11) induced apoptotic cell death, suggesting the presence of a functional Fas receptor. In addition, proteasome inhibitor‐induced apoptosis was prevented by the addition of antagonistic anti‐FasL antibody (4A5) or z‐IETD.fmk, a potent inhibitor of caspase‐8, indicating the involvement of the Fas receptor–ligand apoptotic signaling system in proteasome inhibitor‐mediated apoptosis. Thus, it is suggested that proteasome inhibitors cause the accumulation of c‐Myc protein which induces transiently FasL message to stimulate the Fas receptor–ligand apoptotic signaling pathway.

Brain-specific angiogenesis inhibitor 1 (BAI1) is expressed in human cerebral neuronal cells

Brain-specific angiogenesis inhibitor 1 (BAI1) is a p53-target gene specifically expressed in the brain. We examined the distribution of the endogenous BAI1 protein in normal human brain tissue using a polyclonal antibody against the extracellular region of BAI1. Immunohistochemical study demonstrated that BAI1 was expressed in neuronal cells of the cerebral cortex but not in astrocytes. BAI1 protein was localized in the cellular cytoplasm and membrane. It was predominantly localized in the cellular membrane when expressed in cultured cells by means of gene transfection. BAI1 protein may play an important role in neuronal functions such as synapse formation and signal transduction.

Giant Pituitary Adenoma Manifesting As Homonymous Hemianopia

from a very small sample of the conjunctival lesion. Until several years ago, the management of FL was basically palliative. Recently, the effi cacy of rituximab therapy for FL has been evaluated. In a few clinical trials using rituximab alone for untreated FL, response rates ranged between 61% and 80%. So far, only one case of conjunctival FL treated with rituximab alone has been reported. However, the evidence for diagnosis was not shown, and the follow-up period was only 5 months. Generally, the common side effects of rituximab are not severe, and include fever, chills, and nausea. No side effect of rituximab was observed in this case. Radiotherapy with curative intent for early-stage FL is still questionable because of the recurrence rate of approximately 50% and the treatment side effects. LD-IF-RT (2 × 2 Gy regimen) was initially applied to treat recurrences of advanced FL. Recent studies showed high response rates (>80%) in indolent lymphoma patients. We combined rituximab therapy and LD-IF-RT, since it is too early to evaluate the effi cacy of each of them independently on survival. Minimizing radiation-induced disorders of eyes, that is, cataract, was also another reason to select LD-IFRT. As it happens, this therapeutic idea is supported by a recent study showing that pretreatment with rituximab enhances the radiosensitivity of non-Hodgkin’s lymphoma cells. In conclusion, for early stage conjunctival FL, the combination of anti-CD20 antibody therapy and LD-IF-RT may be an alternative therapy.