Yasuaki Nishimura

Ordering of ceramide formation, caspase activation, and Bax/Bcl-2 expression during etoposide-induced apoptosis in C6 glioma cells

Etoposide (VP-16) a topoisomerase II inhibitor induces apoptosis of tumor cells. The present study was designed to elucidate the mechanisms of etoposide-induced apoptosis in C6 glioma cells. Etoposide induced increased formation of ceramide from sphingomyelin and release of mitochondrial cytochrome c followed by activation of caspase-9 and caspase-3, but not caspase-1. In addition, exposure of cells to etoposide resulted in decreased expression of Bcl-2 with reciprocal increase in Bax protein. z-VAD·FMK, a broad spectrum caspase inhibitor, failed to suppress the etoposide-induced ceramide formation and change of the Bax/Bcl-2 ratio, although it did inhibit etoposide-induced death of C6 cells. Reduced glutathione or N-acetylcysteine, which could reduce ceramide formation by inhibiting sphingomyelinase activity, prevented C6 cells from etoposide-induced apoptosis through blockage of caspase-3 activation and change of the Bax/Bcl-2 ratio. In contrast, the increase in ceramide level by an inhibitor of ceramide glucosyltransferase-1, D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol caused elevation of the Bax/Bcl-2 ratio and potentiation of caspase-3 activation, thereby resulting in enhancement of etoposide-induced apoptosis. Furthermore, cell-permeable exogenous ceramides (C2- and C6-ceramide) induced downregulation of Bcl-2, leading to an increase in the Bax/Bcl-2 ratio and subsequent activation of caspases-9 and -3. Taken together, these results suggest that ceramide may function as a mediator of etoposide-induced apoptosis of C6 glioma cells, which induces increase in the Bax/Bcl-2 ratio followed by release of cytochrome c leading to caspases-9 and -3 activation. Cell Death and Differentiation (2000) 7, 761–772

Influence of Bax or Bcl-2 overexpression on the ceramide-dependent apoptotic pathway in glioma cells

Ceramide has recently been regarded as a potential mediator of apoptosis. In the present study, the effects of Bcl-2 and Bax on the ceramide-mediated apoptotic pathways were examined in glioma cells overexpressing Bcl-2 or Bax. Etoposide, cisplatin and tumor necrosis factor-α induced apoptosis of C6 rat glioma cells which was associated with ceramide formation due to activation of neutral sphingomyelinase, followed by release of mitochondrial cytochrome c into the cytosol and activation of caspases-9 and -3. The growth of C6 cells stably overexpressing either Bcl-2 or Bax was almost equal to that of the vector-transfected cells. Bax overexpression enhanced etoposide-induced apoptosis through acceleration of cytochrome c release and caspases activation. However, Bax had no effect on ceramide formation. Similar findings were obtained in C6 cells and U87-MG human glioblastoma cells which were transiently overexpressed with Bax. In contrast, Bcl-2 overexpression resulted in a retardation of the apoptotic process via prevention of cytochrome c release and caspases activation, and ceramide formation was also blocked when Bcl-2 was highly overexpressed in glioma cells. In addition, transient overexpression of Bcl-xL also exerted inhibitory effects on ceramide formation and apoptotic cell death induced by etoposide. These results indicate that Bax promotes apoptosis regardless of ceramide formation and that Bcl-2 or Bcl-xL prevents ceramide formation by repressing neutral sphingomyelinase as well as ceramide-induced cytochrome c release.

Spinal injuries in snowboarders: risk of jumping as an integral part of snowboarding

BACKGROUND The purpose of this study was to clarify the occurrence rate and characteristics of spinal injuries caused by snowboarding that were sustained at the Okumino skiing area in Gifu Prefecture, Japan, from 1988 to 2000. METHODS This study was a retrospective review of 13,490 cases of snowboard- or ski-related injury treated at Sumi Memorial Hospital over this period. RESULTS A total of 7,188 patients sustained snowboard-related injuries, and 238 of these had spinal injuries caused by snowboarding (3.3%), whereas 6,302 patients sustained ski-related injuries, and 86 of these had spinal injuries caused by skiing (1.4%). Although there were no significant differences in the difficulty of slope, location of vertebral fracture, or spinal cord injury between snowboarders and skiers, the incidence of transverse process fractures was significantly higher in snowboard-related than in skiing-related injury (p < 0.05). In addition, there was a significantly higher incidence of spinal injury among beginner snowboarders than among beginner skiers (p = 0.04). Furthermore, intermediate or expert snowboarders were more likely to be injured because of jumping than beginners (p < 0.001), whereas about 70% of spinal injuries caused by skiing resulted from a simple fall. CONCLUSION Spinal injuries sustained while snowboarding are increasing considerably in incidence and are characterized as complex injuries. We must educate young snowboarders of the risk of this sport, to prevent these serious injuries.

p53 regulates ceramide formation by neutral sphingomyelinase through reactive oxygen species in human glioma cells

The present study was designed to elucidate the relationship between p53 and ceramide, both of which are involved in apoptotic signaling. Treatment of human glioma cells with etoposide caused apoptosis only in cells expressing functional p53. p53 activation was followed by the formation of reactive oxygen species (ROS), superoxide anion (O2−•) measured by hydroethidium oxidation into ethidium and hydrogen peroxide (H2O2) measured by oxidation of 2′,7′-dichlorofluorescin (DCFH) into 2′,7′-dichlorofluorescein (DCF), which was accompanied with ceramide generation through the activation of neutral, but not acid, sphingomyelinase. Superoxide dismutase (SOD), a selective antioxidant for O2−•, had no effects on p53 expression but inhibited ceramide generation and apoptotic cell death caused by etoposide. However, catalase, a specific antioxidant for H2O2, only weakly inhibited and sodium formate, a hydroxyl radical (• OH) scavenger, unaffected etoposide-induced apoptosis. Like etoposide-induced cell death, treatment of glioma cells with the O2−•-releasing agent, pyrogallol, induced typical apoptosis and ceramide generation even in the presence of catalase. In contrast, human glioma cells lacking functional p53, either due to mutation or the expression of E6 protein of human papillomavirus, were highly resistant to etoposide and exhibited no significant change in the ceramide level. Moreover, expression of functional p53 protein in glioma cells expressing mutant p53 using a temperature-sensitive human p53Val138 induced ceramide accumulation by the activation of neutral sphingomyelinase which was dependent on the generation of O2−•. Taken together, these results suggest that p53 may modulate ceramide generation by activation of neutral sphingomyelinase through the formation of O2−•, but not its downstream compounds H2O2 or • OH.

Activation of caspase-9 and -3 during H2O2-induced apoptosis of PC12 cells independent of ceramide formation.

Abstract The treatment of PCI2 cells with H202 (100-500 µM) resulted in typical apoptotic changes including fragmentation and condensation of nuclei, and DINA fragmentation observed as DNA ladder. H2O2-induced apoptosis was associated with activation of caspase-3 as assessed by cleavage of specific fluorogenic substrate peptide and processing of procaspase-3 and poly(ADP-ribose) polymerase. However, formation of ceramide, which often locates upstream of caspase-3, was not observed. The inhibitory peptide relatively specific for caspase-3, z-DEVD-FMK and non-selective caspase inhibitor z-VAD-FMK inhibited activation of caspase-3 and apoptotic cell death. However, the relatively specific inhibitors, Ac-YVKD for caspase-1 and Ac-IETD for caspase-8/6, did not affect the occurrence of apoptotic cell death. As an upstream activation of caspase-3, induction of cytochrome c release followed by processing of procaspase-9 was observed by Western blotting, although the formation of intracellular ceramide was not observed. On the other hand, in PCI2 cells overexpressing Bcl-2, the number of apoptotic cells was markedly decreased and activation of both caspases-9 and -3 was prevented. These results suggest that cytochrome c and caspase-9 initiate the activation of executor caspase-3 in H2O2-treated PCI2 cells, and that Bcl-2 inhibits H2O2-induced release of cytochrome c from mitochondria and then proteolytic processing of procaspase-9. [Neurol Res 2000; 22: 556-564]

Clinical analysis of a series of vertebral aneurysm cases.

We reviewed 38 cases of aneurysms of the vertebral artery treated over the last 10 years: 26 (68%) located at the junction of the vertebral and posterior inferior cerebellar arteries, 10 (26%) at the vertebral artery, and 2 (5%) at the vertebrobasilar union. There were three distinct forms of aneurysms: 20 saccular (53%), 10 fusiform (26%), and 8 dissecting (21%). Among these 38 aneurysms, 33 (87%) had ruptured: 18 of the saccular aneurysms (90%), all 10 of the fusiform aneurysms (100%), and 5 of the dissecting aneurysms (63%). Computed tomography of the 28 ruptured aneurysms revealed diffuse subarachnoid hemorrhage in the basal cistern combined with intraventricular hemorrhage in 24 cases (86%). Magnetic resonance imaging was useful for differentiating between fusiform and dissecting aneurysms. Abnormalities such as a double lumen of the vertebral artery were demonstrated in four of the dissecting aneurysms. The overall surgical results were good for 22 of the 27 surgically treated cases (81%). New bleeding was observed in 8 (24%) of the 33 ruptured aneurysms. The rate of new bleeding was high (60%) in the patients with dissecting aneurysms, and occurred mostly in the acute stage. The incidence of vasospasm was 27%, and only two patients suffered permanent neurological deficits. These findings indicate that the rate of new bleeding tends to be high in patients with saccular and dissecting aneurysms, and thus, they should be treated as early as possible. A preoperative balloon occlusion test should be conducted if proximal occlusion of the vertebral artery is necessary, since proximal occlusion is not always safe, despite angiographic evidence of sufficient contralateral arterial flow.

Changes in the Activity and mRNA Levels of Phospholipase D During Ceramide‐Induced Apoptosis in Rat C6 Glial Cells

Abstract: N‐Acetylsphingosine (C2‐ceramide), a membrane‐permeable analogue, induced apoptosis in C6 glial cells. Phase‐contrast micrographs showed that the round cells appeared 3 h after exposure to 25 µM C2‐ceramide and the number of floating cells increased time‐dependently. Staining with Hoechst 33258 dye showed condensed or fragmented nuclei in round cells at 12 h. DNA fragmentation was also observed by agarose gel electrophoresis at 12 h. To understand the mechanism underlying glial cell death induced by C2‐ceramide treatment, changes in phospholipase D (PLD) activity in response to guanosine 5′‐O‐(3‐thiotriphosphate) (GTPγS) and expression of mRNA levels of PLD isozymes were examined. In cell lysate, GTPγS‐dependent PLD activity was down‐regulated after ceramide treatment in a time‐dependent manner. In the in vitro PLD assay, membrane‐associated PLD activation in response to recombinant ADP‐ribosylation factor 1 was greatly suppressed. Furthermore, levels of rPLD1a and rPLD1b mRNAs were found to be down‐regulated, whereas the level of rPLD2 mRNA increased gradually, peaking at 3 h, followed by a slow decrease, as inferred by reverse transcription‐polymerase chain reaction. Decreases in GTPγS‐dependent PLD activity were well correlated with those in rPLD1a and rPLD1b mRNAs levels. Taken together, these data suggest that levels of PLD enzymes might be decreased by ceramide treatment.

The relationship between Moyamoya disease and bacterial infection

To examine the relationship between Moyamoya disease and bacterial infections, authors studied the serum of 85 cases of Moyamoya disease and the influence of Propionibacterium acnes (P. acnes) infection on intracranial arteries in rats. The serum levels of P. acnes antibody, IgM, transferrin (Tf), alpha 2-macroglobulin (alpha 2M) were significantly higher in Moyamoya disease than in normal volunteers. Moyamoya-like changes of the intracranial internal carotid arteries were histopathologically demonstrated in P. acnes infectious rats. These findings suggest that P. acnes and immunological factors might play a role in the pathogenesis of Moyamoya disease.

Angiotensin I-converting enzyme gene polymorphism in intracranial saccular aneurysm individuals

A polymorphism in the angiotensin I-converting enzyme (ACE) gene has been associated with cerebrovascular diseases as a new potent risk factor. The purpose of this study was to investigate an association of the gene polymorphism with intracranial saccural aneurysmal patients. The study population consisted of 83 aneurysmal patients (age range 41-85 years) (the AN group) and 104 matched control subjects (age range 30-81 years) (the Control group). For detection of the ACE gene polymorphism, the standard PCR method was performed by using genomic DNA isolated from peripheral blood leukocytes. The PCR products were a 490-bp in the presence of the insertion (I) and a 190-bp fragment in the absence of the insertion (D). The ACE gene polymorphism was classified into three genotypes: I/I genotype (a 490-bp band); D/D genotype (a 190-bp band); or I/D genotype (both a 490-bp and a 190-bp band). The number of subjects with I/I, I/D, and D/D genotypes was 38, 40, and 5 in the AN group and 43, 45, and 16 in the Control group, respectively. The frequency of the D/D genotype in the AN group was significantly lower (5/83 = 0.06) than that in the Control group (16/104 = 0.15) (chi 2 = 4.06; p = 0.044). There was no significant difference between the genotype sof hypertensive patients and normotensive patients in the AN group. Thus, this present study suggests that genetic heterogeneity of the ACE gene may be correlated with the etiology of intracranial aneurysms.

THE CHARACTERIZATION OF HUMAN BRAIN TUMOR USING MAGNETIZATION TRANSFER TECHNIQUE IN MAGNETIC RESONANCE IMAGING

The clinical applicability of magnetization transfer (MT) technique in magnetic resonance imaging (MRI) for the estimation of the histological and constitutional feature of brain tumors was investigated. MT effect was evaluated by measuring the MT ratio (MTR). The parameters in 1.5-tesla MRI system were as follows: TR, 50 msec; TE, 5 msec; flip angle, 30 degree; offset frequency of off-resonance MT pulse, 1000 Hz. The sequence was performed in 20 normal volunteers and 45 patients with brain tumors which were characterized histologically and surgically. The MTR for brain tumors was significantly lower than that for normal brain tissue (p < 0.05). The MTR for meningioma was higher than that for the other brain tumors (p < 0.05). In the meningiomas, MTR for fibrous type was higher than that for meningothelial type, but there was no statistical significance. Regarding the physical consistency for the brain tumors, as classified by surgery, there was a statistically significant difference in MTR between the soft tumor group (0.22 +/- 0.03, n = 6) and the hard tumor group (0.36 +/- 0.04, n = 10) (p < 0.01). This study suggested that the MT technique for patients with brain tumor may be useful to understand the characteristics of the tumors presurgically, based on the degree of intermolecular interaction of macromolecule such as protein.