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      Down-Regulation of Bcl-2, Activation of Caspases, and Involvement of Reactive Oxygen Species in 6-Hydroxydopamine-Induced Thymocyte Apoptosis

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          Objective: Our previous work showed that 6-hydroxydopamine (6-OHDA) induced mouse thymocytes to undergo apoptosis both in vivo and in vitro. In the present study, we further investigated the mechanisms of 6-OHDA-induced apoptosis in vitro. Methods: Naive mouse thymocytes were cultured with 6-OHDA. The percentages of apoptotic cells were quantified by propidium iodide staining, and DNA fragmentation was detected by agarose gel electrophoresis. Intracellular Bcl-2 was analyzed by immunofluorescence staining. Cu/Zn superoxide dismutase (Cu/Zn-SOD) activities were measured by the SOD-525 method. Results: The apoptotic effect of 6-OHDA was blocked by desipramine, a catecholamine uptake blocker. Treatment with 6-OHDA caused a reduction in Bcl-2 expression. VAD-FMK, a broad-spectrum caspase inhibitor, and DEVD-CHO, a potent inhibitor of caspase-3, could block 6-OHDA-induced thymocyte apoptosis. However, the specific caspase-1 (ICE) inhibitor YVAD-CMK had no effect. This cell death process was prevented by the protein synthesis inhibitor cycloheximide and by antioxidants. The level of Cu/Zn-SOD activities also decreased after cells were exposed to 6-OHDA. Conclusion: These results suggest an apoptotic effect of 6-OHDA via the uptake of this neurotoxin by thymocytes, and that down-regulation of Bcl-2, activation of caspases, such as caspase-3 but not caspase-1, generation of reactive oxygen species, and new synthesis of proteins are involved in this process.

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          Most cited references 12

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          Glutamate-induced neuronal death: a succession of necrosis or apoptosis depending on mitochondrial function.

          During ischemic brain injury, glutamate accumulation leads to overstimulation of postsynaptic glutamate receptors with intracellular Ca2+ overload and neuronal cell death. Here we show that glutamate can induce either early necrosis or delayed apoptosis in cultures of cerebellar granule cells. During and shortly after exposure to glutamate, a subpopulation of neurons died by necrosis. In these cells, mitochondrial membrane potential collapsed, nuclei swelled, and intracellular debris were scattered in the incubation medium. Neurons surviving the early necrotic phase recovered mitochondrial potential and energy levels. Later, they underwent apoptosis, as shown by the formation of apoptotic nuclei and by chromatin degradation into high and low molecular weight fragments. These results suggest that mitochondrial function is a critical factor that determines the mode of neuronal death in excitotoxicity.
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            Reactive oxygen species regulate activation-induced T cell apoptosis.

            Reactive oxygen species (ROS) mediate apoptosis in a number of cell types. We studied the role that ROS play in activated T cell apoptosis by activating T cells in vivo and then culturing them for a short time. Activated T cells died independently of Fas and TNF alpha. Their death was characterized by rapid loss of mitochondrial transmembrane potential (delta psi(m)), caspase-dependent DNA fragmentation, and superoxide generation. A superoxide dismutase mimetic, Mn (III) tetrakis (5, 10, 15, 20-benzoic acid) porphyrin (MnTBAP), protected T cells from superoxide generation, caspase-dependent DNA loss, loss of delta psi(m), and cell death. These results indicate that ROS can regulate signals involved in caspase activation and apoptosis and may contribute to peripheral T cell deletion.
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              Mitochondria and programmed cell death: back to the future.

               N Zamzami,  P. Petit,  S Susin (1996)
              Programmed cell death, or apoptosis, has in the past few years undoubtedly become one of the most intensively investigated biological processes. However, fundamental questions concerning the molecular and biochemical mechanisms remain to be elucidated. The central question concerns the biochemical steps shared by the numerous death induction pathways elicited by different stimuli. Heterogeneous death signals precede a common effector phase during which cells pass a threshold of 'no return' and are engaged in a degradation phase where they acquire the typical onset of late apoptosis. Alterations in mitochondrial permeability transition linked to membrane potential disruption precede nuclear and plasma membrane changes. In vitro induction of permeability transition in isolated mitochondria provokes the release of a protein factor capable of inducing nuclear chromatin condensation and fragmentation. This permeability transition is regulated by multiple endogenous effectors, including members of the bcl-2 gene family. Inhibition of these effects prevents apoptosis.

                Author and article information

                S. Karger AG
                August 2003
                15 August 2003
                : 10
                : 6
                : 328-336
                aDepartment of Nursing, Chung Hwa College of Medical Technology, Tainan Hsien and Departments of bPharmacology and cMicrobiology and Immunology, National Cheng Kung University, College of Medicine, Tainan, Taiwan, ROC
                71473 Neuroimmunomodulation 2002–03;10:328–336
                © 2003 S. Karger AG, Basel

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                Page count
                Figures: 4, Tables: 2, References: 45, Pages: 9
                Original Paper


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