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Y Enokido

Publications and source records attributed to Y Enokido.

33 records · Page 2Linked to original sources
PubMed1996

Oxygen-induced apoptosis in PC12 cells with special reference to the role of Bcl-2.

We previously reported that PC12h cells are killed by a high oxygen atmosphere. In this study, we further characterized this oxygen-induced cell death and found apoptotic features, as follows. Firstly, chromatin condensation was observed in cells cultured in a 50% O2 atmosphere. Secondly, cycloheximide and cordycepin, protein and RNA synthesis inhibitors, respectively, prevented the oxygen-induced cell death in PC12h cells, suggesting that it is mediated by an intracellular death program. Thirdly, NGF, CPT-cAMP and depolarization by high potassium medium also effectively inhibited this apoptotic cell death in PC12h cells. The effect of high K+ is thought to be mediated by the influx of Ca2+ into cells through voltage-dependent Ca2+ channels, because nifedipine, an L-type Ca2+ channel blocker, inhibited the effect of high K+. In addition, since the oxygen-induced apoptosis was blocked by the antioxidant vitamin E, this oxygen toxicity is suggested to be mediated by reactive oxygen species. To further characterize this oxygen-induced apoptosis at the molecular level, we used PC12 cells overexpressing the proto-oncogene bcl-2. Although a large number of PC12 cells transfected with the control vector died in a 50% O2 atmosphere within 6 days, bcl-2-transfected PC12 cells survived and proliferated. These findings suggested that our system using PC12 cells will be a useful model with which to analyze the molecular mechanisms of apoptosis induced by oxidative stress in neuronal cells.

Animals↗
PubMed1996

Survival factor-insensitive generation of reactive oxygen species induced by serum deprivation in neuronal cells.

To investigate the involvement of reactive oxygen species (ROS) in neuronal apoptosis, we performed confocal and flow cytometric analysis with a ROS-specific fluorogen, 6-carboxy-2', 7'-dichorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Serum deprivation significantly increased the level of ROS in PC12 cells and rat cortical neurons. N,N'-diphenyl-p-phenylenediamine (DPPD), an antioxidant, reduced ROS production induced by serum deprivation and recovered cell survival. However, some survival factors such as nerve growth factor and Bcl-2, which prevented the apoptosis of PC12 cells, did not affect the up-regulation of ROS induced by serum deprivation. Epidermal growth factor which prevented the apoptosis of cortical neurons, did not affect the increase of ROS. These data suggest that survival factors rescue the serum deprivation induced apoptosis independently of ROS production.

Animals↗
PubMed1996

p53 involves cytosine arabinoside-induced apoptosis in cultured cerebellar granule neurons.

The tumor suppressor p53 gene plays a key role in controlling the cell cycle checkpoint and in apoptosis following the exposure of normal cells to DNA damage. To investigate the role of p53 in cytosine arabinoside (Ara C)-induced cell death of CNS neurons, we examined the effect of Ara C on the survival of cultured cerebellar granule neurons from normal wild-type and p53 null mutant mice. When the neurons from wild-type mice were cultured with Ara C, they gradually died after 24 h in culture. In contrast, the neurons from p53 null mutant mice showed a resistance to the Ara C neurotoxicity. These results indicate that p53 involves Ara C-induced apoptosis in cultured cerebellar granule neurons, in which DNA damage may initiate the apoptotic death program of the neurons.

Animals↗
PubMed1996

Free radical-independent protection by nerve growth factor and Bcl-2 of PC12 cells from hydrogen peroxide-triggered apoptosis.

To investigate the mechanism of oxidative stress induced death of PC12 cells, we performed confocal and flow cytometric analysis with a reactive oxygen species (ROS)-specific fluorogen, 6-carboxy-2',7'-dichlorodihydrofluorescein diacetate, di(acetoxymethyl ester) (C-DCDHF-DA). Hydrogen peroxide significantly decreased the number of viable PC12 cells after 24 h. Hydrogen peroxide caused membrane blebbing, nuclear condensation and DNA fragmentation, indicating that the PC12 cells died due to apoptosis. The hydrogen peroxide-triggered apoptosis of PC12 cells was associated with enhanced ROS production in a dose-dependent manner by measuring with C-DCDHF-DA. Nerve growth factor (NGF) and Bcl-2 inhibited the hydrogen peroxide-induced apoptosis of PC12 cells. Neither of them, however, reduced the ROS production in PC12 cells. These data suggest that NGF or Bcl-2 protects PC12 cells from hydrogen peroxide-triggered apoptosis independently from ROS production.

Animals↗
PubMed1996

Involvement of p53 in DNA strand break-induced apoptosis in postmitotic CNS neurons.

The tumour suppressor p53 gene serves as a critical regulator of the cell cycle and of apoptosis following the exposure of normal cells to DNA damage. To examine the role of p53 in postmitotic CNS neurons, we cultured cerebellar neurons from normal wild-type mice and mutant p53-null mice under various conditions inducing neuronal death. When cerebellar neurons from 15- to 16-day postnatal wild-type mice were treated with ionizing radiation or DNA-damaging agents, massive neuron death occurred after 24-72 h. In contrast, neurons from p53-/- mice evidently resisted gamma-irradiation and some DNA-damaging agents, such as etoposide and bleomycin. On the other hand, low-K+ medium-induced apoptosis of cerebellar neurons was not affected by p53 status. Neither cell cycle progression nor DNA synthesis occurred during cell death induced by gamma-irradiation and low-K+ medium, as well as in normal cultures of p53+/+ and p53-/- neurons. These results suggest that p53 is required for the apoptotic death of postmitotic cerebellar neurons induced by DNA strand breaks.

Animals↗
PubMed1995

Flow cytometric analysis of serum deprivation-induced apoptosis of PC12 cells, with special reference to role of bcl-2.

To investigate the mechanism of serum deprivation-induced apoptosis in PC12 cells, we performed flow cytometry with the viable cell-specific fluorogen, fluorescein diacetate (FDA). Intact PC12 cells were positive when stained with FDA, and those cells were identical with the major population which possess larger forward and side scattered light. Apoptotic PC12 cells decreased the forward and side scatter light associated with the loss of FDA-positive cells. Nerve growth factor (NGF) and the bcl-2 protein protected the cells from serum deprivation-induced apoptosis. These data suggested that the process of apoptosis in PC12 cells can be analyzed with flow cytometry.

Animals↗
PubMed1995

Epidermal growth factor prevents oxygen-triggered apoptosis and induces sustained signalling in cultured rat cerebral cortical neurons.

Epidermal growth factor (EGF), a conventional mitogenic factor, acts as a neurotrophic factor on several types of neurons in the central nervous system. We found that EGF prevented the death of rat cerebral cortical neurons cultured in a 50% oxygen atmosphere. This high-oxygen-triggered cell death showed features of apoptotic cell death, which was blocked by inhibitors of RNA or protein synthesis. EGF prevented the oxygen-induced death of the cultured cortical neurons in a dose-dependent manner. Basic fibroblast growth factor (bFGF) also prevented this cell death, although there was no apparent additive effect of EGF and bFGF. Among the cultured cortical neurons, we observed neurons possessing the EGF receptor and cells expressing c-Fos protein in response to EGF. The cortical neurons were cultured in the presence of cytosine arabinoside, and the number of glial fibrillary acidic protein-positive astroglial cells was < 0.5% of that of the corresponding microtubule-associated protein 2-positive neurons. Therefore, the effect of EGF on the cultured cortical neurons is thought to be due to a direct action. We also examined EGF-induced signalling in the cultured cortical neurons. We found that EGF induced the sustained tyrosine phosphorylation of the EGF receptor and sustained the activation of mitogen-activated protein kinase in the cultured cortical neurons. We suggest that EGF may exert the survival effect through the prolonged activation of the EGF signalling.

Animals↗
PubMed1995

Brain-derived neurotrophic factor (BDNF) can prevent apoptosis of rat cerebellar granule neurons in culture.

Cerebellar granule neurons obtained from 9-day-old rats were grown in vitro for 4 days in high K+ (26 mM) medium. The culture medium was then replaced with that containing low K+ (5 mM) which caused a large number of granule neurons to die. The death of granule neurons has been characterized as apoptosis. In this study, we investigated the effects of various neurotrophins on neuronal survival using the above system. We found that brain-derived neurotrophic factor (BDNF) and neurotrophin-4/5 (NT-4/5) but not nerve growth factor (NGF) can protect these neurons from apoptosis in low K+. Neurotrophin-3 (NT-3) had a small effect on neuronal survival as reported. To determine whether the granule neurons respond directly to BDNF, we analyzed the induction of the Fos protein in these neurons. Individual cells that synthesize Fos protein after exposure to neurotrophin can be recognized using antibodies to Fos. Immunocytochemical staining of the cultures demonstrated that a relatively large number of cerebellar granule neurons showed immunoreactivity in response to BDNF, but few of them were immunoreactive in the absence of BDNF or in the presence of NGF. Our results suggested that BDNF has a direct effect on mature cerebellar granule neurons and can protect these neurons from apoptosis in low K+.

Animals↗
PubMed1994

[Neuronal cell death and apoptosis].

Neuronal cell death is thought to be an important phenomenon not only for the developmental but also for the post-matured stages of the PNS and CNS neurons. In the developmental stages, massive neuronal death is observed at specific periods, e.g. the immediately after the arrival of axons to the postsynaptic target fields. This phenomenon is precisely programmed in the developing nervous system, so that they are called physiological or naturally occurring neuronal death. It has been shown that this programmed neuronal death is mediated by apoptotic death cascade, as follows: 1) neurotrophic agents, such as nerve growth factor, or afferent electrical input prevent neuronal death; 2) the macromolecular synthesis inhibitors, such as cycloheximide or actinomycin-D, prevent cell death; 3) DNA fragmentation is detected during the course of cell death; 4) variations of apoptotic morphology are observed in programmed neuronal death. Furthermore, recent studies also suggest that such apoptotic neuronal death occurs in the matured nervous system, e.g. neuro-degenerative diseases, such as Alzheimer's and Parkinson's diseases, or aging. Thus, it is expected that the study of apoptosis in the nervous system give us useful clues to know how the neuronal network are constructed in the development and why the neurodegenerative disease and brain aging occur.

Alzheimer Disease↗
PubMed1993

Apoptotic cell death occurs in hippocampal neurons cultured in a high oxygen atmosphere.

When embryonic rat hippocampal neurons were cultured in a 50% oxygen atmosphere, neurons gradually died after 20 h in culture. This death pattern was found to be mediated by an intracellular active death program, so called apoptosis, as follows: (1) Cycloheximide and actinomycin-D, protein and RNA synthesis inhibitors, respectively, prevented cell death, indicating that cell death required new protein biosynthesis. (2) DNA fragmentation (called a "DNA ladder"), a specific biochemical marker of apoptosis, was detected during the course of cell death. (3) Depolarization with high K+ medium (26-50 mM) prevented cell death. This effect was suppressed by some dihydropyridine derivatives, L-type Ca channel blockers, such as nifedipine and nicardipine. These results indicate that increased levels of oxygen activate an apoptotic mechanism in the cultured hippocampal neurons, and suggest that neuronal activity may protect the neurons from oxygen-induced apoptosis.

Animals↗
PubMed1993

In vitro model of hypoxia: basic fibroblast growth factor can rescue cultured CNS neurons from oxygen-deprived cell death.

We established an in vitro hypoxia model and investigated the protective effect of basic fibroblast growth factor (bFGF) against neuronal cell death caused by hypoxia. Hippocampal neurons obtained from rats on embryonic day (E) 17 and 20 and on postnatal day (P) 4 were cultured for 6-24 h in an oxygen-deprived state. This in vitro hypoxia study showed that the cultured neurons were sensitive to the oxygen deprivation. The cultured P4 rat hippocampal neurons seemed to be weaker in the hypoxia condition than those of E17 and E20 rats, suggesting that the cultured postnatal cells might be sensitive to hypoxia. bFGF, but not nerve growth factor, prevented the neuronal cell death caused by hypoxia in a dose-dependent manner.

Animals↗
PubMed1992

Basic fibroblast growth factor rescues CNS neurons from cell death caused by high oxygen atmosphere in culture.

In the present study, we cultured rat CNS neurons and tested the neurotrophic support provided by basic fibroblast growth factor (bFGF) to prevent the oxygen-induced neuronal cell death. When rat basal forebrain (septum and vertical limb of diagonal band of Broca) cells of embryonic day 20 were cultured in a serum-free medium containing 5 microM cytosine arabinoside in a 50% oxygen atmosphere, the neuronal cells, which were immunostained by an anti-microtubule-associated protein 2 (MAP2) antibody, gradually died after 1 day in culture. After 3.5 days in culture, only 2-5% of neuronal cells survived. This oxygen-induced cell death of cultured basal forebrain neurons was reversed by the addition of bFGF at a concentration of 100 ng/ml. This cell-saving effect was dose-dependent, and the ED50 value was 12 ng/ml. Nerve growth factor (NGF) and insulin-like growth factor II could not prevent cell death. The activity of choline acetyltransferase was also maintained when bFGF was present in the basal forebrain culture. Viable astroglial cells, which were immunostained by an anti-glial fibrillary acidic protein, accounted for a few percent of the total number of cells after 3 days in culture both with and without 100 ng/ml of bFGF. The survival-enhancing effect of bFGF was observed not only in basal forebrain neurons but also in neocortical and hippocampal neurons. However, the sensitivity to oxygen toxicity of cultured neurons from the 3 CNS regions varied greatly. The neocortical neurons were the most sensitive to oxidative stress, while the hippocampal neurons were the most resistant. These results suggest that bFGF plays an important role in saving neuronal cells from oxidative stress during their long life without division.

Animals↗
PubMed1990

High oxygen atmosphere for neuronal cell culture with nerve growth factor. I. Primary culture of basal forebrain cholinergic neurons from fetal and postnatal rats.

Cholinergic neurons cultured from postnatal days 11-13 (P11-P13) rat basal forebrain showed better survival in the culture condition using a 50% O2 atmosphere with and without nerve growth factor (NGF) than in a low (10 or 20%) O2 atmosphere. Except for the culture at a low cell density, the beneficial effect of the highly oxidized culture condition was found in the culture from P3 neurons, but not from embryonic day 18 neurons. The survival of microtubule-associated protein 2 (MAP2)-positive neurons in culture from P3 basal forebrain regions was more enhanced in a 50% O2 atmosphere than in 20% and also 10% O2 atmosphere. The viable number of the MAP2-positive neurons in a 10% O2 condition was about half of that in a 20% condition. These results suggest that the response of the cultured neurons to an incubator O2 concentration changes during the neuronal development in CNS from fetal to postnatal stages.

Acetylcholine↗
PubMed1990

High oxygen atmosphere for neuronal cell culture with nerve growth factor. II. Survival and growth of clonal rat pheochromocytoma PC12h cells.

When clonal rat pheochromocytoma PC12h cells were cultured in a 50% O2 atmosphere, cells gradually died during the cultivation. On the other hand, the addition of NGF at the final concentration of 50 ng/ml could rescue the cells from death. The culture in a 40% O2 atmosphere had little effect on the growth of PC12h cells, as compared with the culture in a normal 20% O2 condition. A very high O2 concentration, as 60%, caused severe damage to PC12h cell growth, and the restoration of cell growth by NGF seemed to be insufficient. PC12h cells were fully differentiated and extended dense long neurites by NGF even in a 50% O2 atmosphere. However, the neurite extension in the culture in a 60% O2 atmosphere was suppressed. The cell-saving effect of NGF on cell death in culture under a 50% O2 atmosphere was dose-dependent, and the ED50 value of NGF was 5 ng/ml. Basic fibroblast growth factor and epidermal growth factor also had a potent effect to rescue the cell death in the high O2 culture, but insulin had no effect. Since the differentiation effects of NGF on PC12h cells are thought to offer a model system to investigate the effect of NGF on neurons, the present observations suggest that a protection machinery for high O2 toxicity to neurons may exist in the neuronal differentiated PC12h cells by NGF, but not in the undifferentiated cells.

Animals↗