acetyl-aspartyl-glutamyl-valyl-aspartal and Neuroblastoma

While it has been reported that familial Alzheimer's disease (FAD)-linked mutants of amyloid precursor protein (APP) and presenilin (PS)2 induce neuronal cytotoxicity in a manner sensitive to antioxidant and pertussis toxin (PTX), little of the mechanism for PS1-mediated neuronal cell death has been characterized. We previously found that multiple mechanisms, different in detail, underlie cytotoxicities by two FAD-linked mutants of APP, using neuronal cells with an ecdysone-controlled expression system. Here we report that this system revealed that (i) low expression of FAD-linked M146L-PS1 caused neuronal cell death, whereas that of wild-type (wt)PS1 did not; (ii) mutation-specific cytotoxicity by M146L-PS1 was sensitive to antioxidant glutathione-ethyl-ester and resistant to Ac-DEVD-CHO; (iii) cytotoxicity by higher expression of wtPS1 was resistant to both; and (iv) cytotoxicity by M146L-PS1 was inhibited by PTX. It was also highly likely that the involved superoxide-generating enzyme was nitric oxide synthase (NOS), and that the PTX-sensitive cytotoxic signal by M146L-PS1 was mediated by none of the G(i/o) proteins. We conclude that M146L-PS1 activates a NOS-mediated cytotoxic pathway via a novel PTX target.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Cell Death; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Gene Expression; Glutathione; GTP-Binding Proteins; Membrane Proteins; Mice; Neuroblastoma; Neurons; Neurotoxins; NG-Nitroarginine Methyl Ester; Nitric Oxide Synthase; Oligopeptides; omega-N-Methylarginine; Pertussis Toxin; Presenilin-1; Radiation-Protective Agents; Rats; Tumor Cells, Cultured; Virulence Factors, Bordetella

Continuous and long-lasting exposure to tert-butylhydroperoxide (t-BOOH) increased the number of apoptotic SH-SY5Y human neuroblastoma cells both in the presence and in the absence of the intracellular Ca(2+) ion chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). In addition, t-BOOH exposure induced activation of CPP32, as demonstrated by poly-(ADP-ribose) polymerase (PARP) cleavage, and of ICH-1L caspases. Exposure to t-BOOH also induced a time-dependent release of cytochrome c. Interestingly, in the presence of BAPTA, CPP32 activation still occurred, whereas ICH-1L activation was blocked. Ac-DEVD-CHO, an inhibitor of CPP32 activity, prevented the appearance of apoptotic cells, whereas the inhibitor of ICH-1L activity Z-VDVAD-FMK did not. Collectively, these findings demonstrate that in SH-SY5Y neuroblastoma cells exposure to continuous and long-lasting oxidative stress induced activation of caspase-3 that was independent of intracellular Ca(2+) ion concentration ([Ca(2+)](i)) elevation but led to cell apoptosis. In contrast, caspase-2 activation was dependent on [Ca(2+)](i) increase but did not result in apoptosis.

Topics: Apoptosis; Blotting, Western; Calcium; Caspase 2; Caspase 3; Caspase Inhibitors; Caspases; Cell Line; Egtazic Acid; Enzyme Inhibitors; Free Radicals; Humans; In Situ Nick-End Labeling; Neuroblastoma; Oligopeptides; Oxidative Stress; tert-Butylhydroperoxide; Time Factors; Tumor Cells, Cultured

Dlk/ZIP kinase is a member of the Death Associated Protein (DAP) kinase family of pro-apoptotic serine/threonine kinases that have been implicated in regulation of apoptosis and tumour suppression. Expression of both Dlk/ZIP kinase and its interaction partner Par-4 is maintained in four medulloblastoma cell lines investigated, whereas three of seven neuroblastoma cell lines have lost expression of Par-4. Overexpression of a constitutively pro-apoptotic deletion mutant of Dlk/ZIP kinase induced significant apoptosis in D283 medulloblastoma cells. Cell death was characterized by apoptotic membrane blebbing, and a late stage during which the cells had ceased blebbing and were drastically shrunken or disrupted into apoptotic bodies. Over-expression of the anti-apoptotic Bcl-xL protein had no effect on Dlk/ZIP kinase-induced membrane blebbing, but potently inhibited Dlk/ZIP kinase-induced cytochrome c release and transition of cells to late stage apoptosis. Treatment with caspase inhibitors delayed, but did not prevent entry into late stage apoptosis. These results demonstrate that Dlk/ZIP kinase-triggered apoptosis involves the mitochondrial apoptosis pathway. However, cell death proceeded in the presence of caspase inhibitors, suggesting that Dlk/ZIP kinase is able to activate alternative cell death pathways. Alterations of signal transduction pathways leading to Dlk/ZIP kinase induced apoptosis or loss of expression of upstream activators could play important roles in tumour progression and metastasis of neural tumours.

Topics: Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Calcium-Calmodulin-Dependent Protein Kinases; Carrier Proteins; Caspase Inhibitors; Cysteine Proteinase Inhibitors; Cytochrome c Group; Death-Associated Protein Kinases; Gene Expression Regulation, Neoplastic; Green Fluorescent Proteins; Humans; Intracellular Membranes; Intracellular Signaling Peptides and Proteins; Luminescent Proteins; Medulloblastoma; Microscopy, Fluorescence; Mitochondria; Mutation; Neuroblastoma; Oligopeptides; Protein Serine-Threonine Kinases; Proto-Oncogene Proteins c-bcl-2; Recombinant Fusion Proteins; Signal Transduction; Transfection; Tumor Cells, Cultured

Fenretinide is an effective inducer of apoptosis in many malignancies but its precise mechanism(s) of action in the induction of apoptosis in neuroblastoma is unclear. To characterize fenretinide-induced apoptosis, neuroblastoma cell lines were treated with fenretinide and flow cytometry was used to measure apoptosis, free radical generation, and mitochondrial permeability changes. Fenretinide induced high levels of caspase-dependent apoptosis accompanied by an increase in free radicals and the release of cytochrome c in the absence of mitochondrial permeability transition. Apoptosis was blocked by two retinoic acid receptor (RAR)-beta/gamma-specific antagonists, but not by an RARalpha-specific antagonist. Free radical induction in response to fenretinide was not blocked by the caspase inhibitor ZVAD or by RAR antagonists and was only marginally reduced in cells selected for resistance to fenretinide. Therefore, free radical generation may be only one of a number of intracellular mechanisms of apoptotic signaling in response to fenretinide. These results suggest that the effector pathway of fenretinide-induced apoptosis of neuroblastoma is caspase dependent, involving mitochondrial release of cytochrome c independently of permeability changes, and mediated by specific RARs. As the mechanism of action of fenretinide may be different from other retinoids, this compound may be a valuable adjunct to neuroblastoma therapy with retinoic acid and conventional chemotherapeutic drugs.

Topics: Amino Acid Chloromethyl Ketones; Antineoplastic Agents; Apoptosis; Caspase Inhibitors; Caspases; Cell Membrane Permeability; Cysteine Proteinase Inhibitors; Cytochrome c Group; Fenretinide; Free Radicals; Humans; Mitochondria; Neuroblastoma; Oligopeptides; Poly(ADP-ribose) Polymerases; Receptors, Retinoic Acid; Tumor Cells, Cultured

We have attempted to elucidate the precise mechanism of nitric oxide (NO)-induced apoptotic neuronal cell death. Enzymatic cleavages of DEVD-AFC, VDVAD-AFC, and LEHD-AFC (specific substrates for caspase-3-like protease (caspase-3 and -7), caspase-2, and caspase-9, respectively) were observed by treatment with NO. Western blot analysis showed that pro-forms of caspase-2, -3, -6, and -7 are decreased during apoptosis. Interestingly, Ac-DEVD-CHO, a caspase-3-like protease inhibitor, blocked not only the decreases in caspase-2 and -7, but also the formation of p17 from p20 in caspase-3 induced by NO, suggesting that caspase-3 exists upstream of caspase-2 and -7. Bongkrekic acid, a potent inhibitor of mitochondrial permeability transition, specifically blocked both the loss of mitochondrial membrane potential and subsequent DNA fragmentation in response to NO. Thus, NO results in neuronal apoptosis through the sequential loss of mitochondrial membrane potential, caspase activation, and degradation of inhibitor of caspase-activated DNase (CAD) (CAD activation).

Topics: Apoptosis; Bongkrekic Acid; Caspase 2; Caspase 3; Caspase 7; Caspase 9; Caspases; Coumarins; DNA Fragmentation; Enzyme Activation; Humans; Intracellular Membranes; Membrane Potentials; Mitochondria; Neuroblastoma; Nitric Oxide; Oligopeptides; Serine Proteinase Inhibitors; Substrate Specificity; Tumor Cells, Cultured