benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and lactacystin
benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone has been researched along with lactacystin* in 14 studies
Other Studies
14 other study(ies) available for benzyloxycarbonylvalyl-alanyl-aspartyl-fluoromethyl-ketone and lactacystin
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Post-translational modification of cyclin A1 is associated with staurosporine and TNFalpha induced apoptosis in leukemic cells.
Understanding of molecular mechanisms underlying the effects of cell cycle proteins in response to the chemotherapeutic agents is of great importance for improving the efficacy of targeted therapeutics and overcoming resistance to chemotherapeutic agents. Staurosporine and tumor necrosis factor alpha (TNFalpha) are the therapeutic agents that inhibit tumor cell growth by inducing cell death. Staurosporine induces apoptosis through the intrinsic pathway, while TNFalpha trigger the cell death via the extrinsic apoptotic pathway. We have previously demonstrated that the cell cycle regulatory protein, cyclin A1 played an important role in the development of acute myeloid leukemia (AML), and cyclin A1 expression correlated with disease characteristics and patient outcome in leukemia. However, it remains unknown how cyclin A1 expression is regulated in leukemic cells treated with the therapeutic agents. Here, we demonstrate that cyclin A1 protein is regulated by proteasome-mediated ubiquitination and degradation in untreated U-937 cells. Interestingly, ubiquitination- and proteasomal-mediated degradation of cyclin A1 is prevented in cells treated with staurosporine or TNFalpha. Induction of apoptosis in U-937 cells by staurosporine or TNFalpha resulted in an increase in cyclin A1 protein expression, which correlated well with cyclin A1 protein modification and the activation of caspase-3. Blocking caspases activity by Z-VAD-FMK had no effect on the increased cyclin A1 expression, suggesting that cyclin A1 might be regulated by caspase-3 independent pathways. We further propose that CDC25C may be associated with cyclin A1 protein modification in response to staurosporine or TNFalpha treatment. Our results suggest that cyclin A1 protein is stabilized via post-transcriptional modification in response to apoptosis induced by staurosporine or TNFalpha. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Caspase 3; Caspase Inhibitors; cdc25 Phosphatases; Cell Line, Tumor; Cyclin A1; Cyclin-Dependent Kinase 2; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Humans; Leukemia; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Processing, Post-Translational; Staurosporine; Tumor Necrosis Factor-alpha; Ubiquitination | 2009 |
Involvement of the ubiquitin pathway in decreasing Ku70 levels in response to drug-induced apoptosis.
Ku70 plays an important role in DNA damage repair and prevention of cell death. Previously, we reported that apoptosis caused a decrease in cellular Ku70 levels. In this study, we analyzed the mechanism of how Ku70 levels decrease during drug-induced apoptosis. In HeLa cells, staurosporin (STS) caused a decrease in Ku70 levels without significantly affecting Ku70 mRNA levels. We found that Ku70 protein was highly ubiquitinated in various cell types, such as HeLa, HEK293T, Dami (a megakaryocytic cell line), endothelial, and rat kidney cells. An increase in ubiquitinated Ku70 protein was observed in apoptotic cells, and proteasome inhibitors attenuated the decrease in Ku70 levels in apoptotic cells. These results suggest that the ubiquitin-proteasome proteolytic pathway plays a role in decreasing Ku70 levels in apoptotic cells. Ku70 forms a heterodimer with Ku80, which is required for the DNA repair activity of Ku proteins. We also found that Ku80 levels decreased in apoptotic cells and that Ku80 is a target of ubiquitin. Ubiquitinated Ku70 was not found in the Ku70-Ku80 heterodimer, suggesting that modification by ubiquitin inhibits Ku heterodimer formation. We propose that the ubiquitin-dependent modification of Ku70 plays an important role in the control of cellular levels of Ku70. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antigens, Nuclear; Apoptosis; Caspase Inhibitors; Cell Line; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Doxorubicin; Gene Expression; HeLa Cells; Humans; Ku Autoantigen; Leupeptins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Signal Transduction; Staurosporine; Ubiquitin; Ubiquitin-Protein Ligase Complexes | 2006 |
Induction of apoptosis in human osteosarcoma Saos-2 cells by the proteasome inhibitor MG132 and the protective effect of pRb.
Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Antioxidants; Apoptosis; bcl-X Protein; Blotting, Western; Caspase 3; Caspase 8; Caspases; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cytochromes c; Dose-Response Relationship, Drug; Flow Cytometry; Humans; Leupeptins; Membrane Potentials; Microscopy, Fluorescence; Mitochondria; Multienzyme Complexes; Osteosarcoma; Protease Inhibitors; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Reactive Oxygen Species; Retinoblastoma Protein; Time Factors; Transfection; Tumor Suppressor Protein p53 | 2003 |
c-Jun N-terminal kinase pathway mediates Lactacystin-induced cell death in a neuronal differentiated Neuro2a cell line.
The ubiquitin-proteasome pathway is an intracellular protein degradation pathway responsible for degradation of many regulatory proteins that must be rapidly eliminated normally. Some recent studies reported that a proteasome dysfunction was involved in the pathogenesis of neurodegenerative diseases. Thus, there is now considerable interest in the possible role of proteasome in this regard. Here we show that inhibition of proteasomal function by Lactacystin-induced cell death in a neuronal differentiated Neuro2a (nN2a) cell line but not in an undifferentiated Neuro2a (N2a) cell line. Cell death was accompanied by both the activation of c-Jun N-terminal kinase, p38 and caspase-3. A pan-caspase inhibitor, Z-VAD-FMK, or SB203580, a p38 inhibitor could not inhibit cell death induced by Lactacystin, whereas nN2a cell lines with stable expression of the dominant negative mutant of c-Jun N-terminal kinase showed a remarkable suppression of cell death. Lactacystin-induced cell death is mediated through the c-Jun N-terminal kinase pathway but not the caspase-dependent pathway in a nN2a cell line. Our results shed light on the association among the proteasomal dysfunction, JNK pathway and neuronal cell death, leading to the elucidation of its possible role in the pathogenesis of neurodegenerative diseases. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Caspase 3; Caspase Inhibitors; Caspases; Cell Death; Cell Nucleus; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Enzyme Activation; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; Multienzyme Complexes; Neurons; p38 Mitogen-Activated Protein Kinases; Proteasome Endopeptidase Complex; Tumor Cells, Cultured | 2002 |
Proteasomes modulate balance among proapoptotic and antiapoptotic Bcl-2 family members and compromise functioning of the electron transport chain in leukemic cells.
The mechanism underlying apoptosis induced by proteasome inhibition in leukemic Jurkat and Namalwa cells was investigated in this study. The proteasome inhibitor lactacystin differentially regulated the protein levels of proapoptotic Bcl-2 family members and Bik was accumulated at the mitochondria. Bik overexpression sufficed to induce apoptosis in these cells. Detailed examination along the respiration chain showed that lactacystin compromised a step after complex III, and exogenous cytochrome c could overcome this compromise. Probably as a result, the succinate-stimulated generation of mitochondrial membrane potential was significantly diminished. Bcl-x(L) interacted with Bik in the cells, and Bcl-x(L) overexpression prevented cytochrome c leakage out of the mitochondria, corrected the mitochondrial membrane potential defect, and protected the cells from apoptosis. These results show that proteasomes can modulate apoptosis of lymphocytes by affecting the half-life of Bcl-2 family members, Bik being one of them. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Apoptosis Regulatory Proteins; bcl-X Protein; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytochrome c Group; Electron Transport; Enzyme Activation; Humans; Intracellular Membranes; Jurkat Cells; Leukemia, B-Cell; Leukemia, T-Cell; Membrane Potentials; Membrane Proteins; Mitochondria; Mitochondrial Proteins; Multienzyme Complexes; Peptide Hydrolases; Permeability; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Tumor Cells, Cultured | 2001 |
Proteasome inhibitor-induced apoptosis of B-chronic lymphocytic leukaemia cells involves cytochrome c release and caspase activation, accompanied by formation of an approximately 700 kDa Apaf-1 containing apoptosome complex.
Proteasome inhibitors, including lactacystin and MG132 (carbobenzoxyl-leucinyl-leucinyl-leucinal), potently induce apoptosis in leukaemic B cells from patients with B cell chronic lymphocytic leukaemia (B-CLL). This pro-apoptotic effect occurs in cells from patients at all stages of the disease, including those resistant to conventional chemotherapy, suggesting that proteasome inhibitors may be useful for treatment of B-CLL. Following initial inhibition of proteasomal activity, these agents induce mitochondrial cytochrome c release and caspase-dependent apoptosis, involving cleavage/activation of caspases -2, -3, -7, -8 and -9. Pre-treatment with the cell permeable caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp (OMe)fluoromethyl ketone (Z-VAD.fmk), did not prevent the release of cytochrome c or partial processing of caspase-9 but prevented activation of effector caspases and the induction of apoptosis. These results suggest that the release of cytochrome c is caspase independent and that caspase-9 is the initiator caspase in proteasome inhibitor-induced apoptosis of B-CLL cells. Activation of B-CLL lysates with dATP results in the formation of an approximately 700 kDa caspase-activating apoptosome complex containing Apaf-1. We describe for the first time the formation of a similar approximately 700 kDa caspase-activating apoptosome complex in B-CLL cells induced to undergo apoptosis by proteasome inhibitors. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptotic Protease-Activating Factor 1; Blotting, Western; Caspase 9; Caspases; Cysteine Endopeptidases; Cytochrome c Group; Cytosol; Enzyme Activation; Humans; Leukemia, Lymphocytic, Chronic, B-Cell; Leupeptins; Microscopy, Electron; Molecular Weight; Multienzyme Complexes; Proteasome Endopeptidase Complex; Protein Biosynthesis; Proteins; Tumor Cells, Cultured | 2001 |
Proteasomal inhibition leads to formation of ubiquitin/alpha-synuclein-immunoreactive inclusions in PC12 cells.
Proteasomal dysfunction has been recently implicated in the pathogenesis of several neurodegenerative diseases, including Parkinson's disease and diffuse Lewy body disease. We have developed an in vitro model of proteasomal dysfunction by applying pharmacological inhibitors of the proteasome, lactacystin or ZIE[O-tBu]-A-leucinal (PSI), to dopaminergic PC12 cells. Proteasomal inhibition caused a dose-dependent increase in death of both naive and neuronally differentiated PC12 cells, which could be prevented by caspase inhibition or CPT-cAMP. A percentage of the surviving cells contained discrete cytoplasmic ubiquitinated inclusions, some of which also contained synuclein-1, the rat homologue of human alpha-synuclein. However the total level of synuclein-1 was not altered by proteasomal inhibition. The ubiquitinated inclusions were present only within surviving cells, and their number was increased if cell death was prevented. We have thus replicated, in this model system, the two cardinal pathological features of Lewy body diseases, neuronal death and the formation of cytoplasmic ubiquitinated inclusions. Our findings suggest that inclusion body formation and cell death may be dissociated from one another. Topics: Acetylcysteine; alpha-Synuclein; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Cell Differentiation; Cyclic AMP; Cysteine Endopeptidases; Immunoblotting; Immunohistochemistry; Inclusion Bodies; Lewy Body Disease; Multienzyme Complexes; Nerve Tissue Proteins; Neuroprotective Agents; Oligopeptides; Parkinson Disease; PC12 Cells; Protease Inhibitors; Proteasome Endopeptidase Complex; Rats; Synucleins; Ubiquitins | 2001 |
JNK (c-Jun N-terminal kinase) and p38 activation in receptor-mediated and chemically-induced apoptosis of T-cells: differential requirements for caspase activation.
Activation of the stress-activated mitogen-activated protein kinases (MAP kinases), c-Jun N-terminal kinase (JNK) and p38, is necessary for the induction of apoptosis in neuronal cells; however, in other cell types their involvement may be stimulus-dependent. In the present study we investigate the activation of JNK and p38 in a single non-neuronal cell type, undergoing receptor-mediated (tumour necrosis factor-related apoptosis-inducing ligand and CD95) or chemically-induced (lactacystin) apoptosis. In Jurkat T-cells, receptor-mediated and chemically-induced apoptosis resulted in a time-dependent activation of the initiator caspases-8 and -9, respectively. Both types of stimuli resulted in a significant activation of JNK and p38, which closely paralleled the time-dependent induction of apoptosis. The caspase inhibitor, benzyloxycarbonyl-Val-Ala-Asp-(OMe) fluoromethyl ketone (z-VAD.FMK) inhibited receptor-mediated apoptosis and suppressed JNK and p38 activation. In contrast, inhibition of lactacystin-induced apoptosis with z-VAD.FMK, as assessed by phosphatidylserine exposure and poly(ADP-ribose) polymerase cleavage, did not inhibit activation of JNK or p38, demonstrating that during chemically-induced apoptosis, activation of JNK and p38 is independent of effector caspases. The role of p38 in apoptosis was assessed using the specific p38 inhibitor, SB203580. No effect on the induction of apoptosis or caspase activation was observed, although activation of mitogen-activated protein kinase-activated protein kinase-2 (MAPKAPK-2), an immediate downstream target of p38, was inhibited. Therefore neither p38 activation nor activation of MAPKAPK-2 is critical for induction of either receptor- or chemically-induced apoptosis. Thus, within a single cell type, (1) the mechanism of p38 and JNK activation during apoptosis is stimulus-dependent and (2) activation of the p38 pathway is not required for caspase activation or apoptosis, assessed by phosphatidylserine exposure, but may still be required to elicit other features of the apoptotic phenotype. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Apoptosis Regulatory Proteins; Caspase 3; Caspase 8; Caspase 9; Caspase Inhibitors; Caspases; Cysteine Proteinase Inhibitors; Enzyme Activation; Enzyme Inhibitors; fas Receptor; Humans; Imidazoles; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Jurkat Cells; Membrane Glycoproteins; Mitogen-Activated Protein Kinases; p38 Mitogen-Activated Protein Kinases; Protein Serine-Threonine Kinases; Pyridines; T-Lymphocytes; Time Factors; TNF-Related Apoptosis-Inducing Ligand; Tumor Necrosis Factor-alpha | 2000 |
Decreased levels of proteasome activity and proteasome expression in aging spinal cord.
Neuron death and neuron degeneration occur in the CNS during the course of aging. Although multiple cellular alterations transpire during the aging process, those that mediate age-associated neuron death have not been identified. Recent evidence implicates oxidative stress as a possible means of neuron death and neuron degeneration during aging. In the present study, we demonstrate a marked decrease in multicatalytic proteasome activity in the spinal cord of Fisher 344 rats at 12, 24 and 28 months, compared with spinal cord tissue from 3-week- and 3-month-old animals. Application of oxidative injury (FeSO(4)) or the lipid peroxidation product 4-hydroxynonenal decreases multicatalytic proteasome activity in a time- and dose-dependent manner in a motor neuron cell line. Loss of multicatalytic proteasome activity occurs before the loss of multicatalytic proteasome immunoreactivity, with FeSO(4)- and 4-hydroxynonenal-mediated decreases ameliorated by the application of a cell permeable form of the antioxidant glutathione. Application of multicatalytic proteasome inhibitors, but not inhibitors of lysosomal proteases, induced neuron death that was attenuated by the caspase inhibitors benzyloxycarbonyl-Val-Ala-Asp-(O-methyl) fluoromethyl ketone or N-acetyl-Asp-Glu-Val-Asp-Cho (aldehyde). Together, these data suggest that multicatalytic proteasome inhibition occurs during aging of the spinal cord, possibly as the result of oxidative stress, and that multicatalytic proteasome inhibition may be causally related to neuron death. Topics: Acetylcysteine; Aging; Aldehydes; Amino Acid Chloromethyl Ketones; Animals; Cell Death; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Glutathione; Iron; Lipid Peroxidation; Lysosomes; Mice; Motor Neurons; Multienzyme Complexes; Neuroblastoma; Oligopeptides; Oxidative Stress; Proteasome Endopeptidase Complex; Rats; Rats, Inbred F344; Reactive Oxygen Species; Spinal Cord; Tumor Cells, Cultured | 2000 |
Protease inhibitor-induced apoptosis: accumulation of wt p53, p21WAF1/CIP1, and induction of apoptosis are independent markers of proteasome inhibition.
Inhibitors of proteases are currently emerging as a potential anti-cancer modality. Nonselective protease inhibitors are cytotoxic to leukemia and cancer cell lines and we found that this cytotoxicity is correlated with their potency as inhibitors of the proteasome but not as inhibitors of calpain and cathepsin. Highly selective inhibitors of the proteasome were more cytotoxic and fast-acting than less selective inhibitors (PS341>>ALLN>>ALLM). Induction of wt p53 correlated with inhibition of the proteasome and antiproliferative effect in MCF7, a breast cancer cell line, which was resistant to apoptosis caused by proteasome inhibitors. In contrast, inhibitors of the proteasome induced apoptosis in four leukemia cell lines lacking wt p53. The order of sensitivity of leukemia cells was: Jurkat>HL60> or =U937>>K562. The highly selective proteasome inhibitor PS-341 induced cell death with an IC50 as low as 5 nM in apoptosis-prone leukemia cells. Cell death was preceded by p21WAF1/CIP1 accumulation, an alternative marker of proteasome inhibition, and by cleavage of PARP and Rb proteins and nuclear fragmentation. Inhibition of caspases abrogated PARP cleavage and nuclear fragmentation and delayed, but did not completely prevent cell death caused by PS-341. Reintroduction of wt p53 into p53-null PC3 prostate carcinoma cells did not increase their sensitivity to proteasome inhibitors. Likewise, comparison of parental and p21-deficient cells demonstrated that p21WAF1/CIP1 was dispensable for proteasome inhibitor-induced cytotoxicity. We conclude that accumulation of wt p53 and induction of apoptosis are independent markers of proteasome inhibition. Topics: Acetylcysteine; Acrylates; Amino Acid Chloromethyl Ketones; Apoptosis; Boronic Acids; Bortezomib; Calpain; Cathepsins; Cell Division; Cyclin-Dependent Kinase Inhibitor p21; Cyclins; Cysteine Endopeptidases; Drug Synergism; Genes, p53; Humans; Jurkat Cells; Leupeptins; Multienzyme Complexes; Neoplasm Proteins; Neoplasms; Oligopeptides; Protease Inhibitors; Proteasome Endopeptidase Complex; Pyrazines; Tumor Cells, Cultured; Tumor Suppressor Protein p53; U937 Cells | 2000 |
Caspase and proteasome activity during staurosporin-induced apoptosis in lens epithelial cells.
To determine what caspases are activated during staurosporin-induced apoptosis in cultured bovine lens epithelial cells (BLECs), to study the time course of caspase activation in relation to morphologic changes, and to investigate the effect of caspase and/or proteasome inhibition on apoptosis.. BLECs were incubated with staurosporin at different concentrations or for different times. Phosphatidylserine (PS) externalization was detected by annexin-V labeling, nuclear morphology was studied by staining with Hoechst 33342 stain (Hoechst, Frankfurt, Germany), and the percentage of apoptotic cells was determined by the TdT-dUTP terminal nick-end labeling (TUNEL) assay. The activity of caspase-1, -2, -3, -4, -8, and -9 as well as the chymotrypsin-like activity of the proteasome was measured by the use of fluorogenic peptide substrates. Inhibition of the proteasome was performed by incubation with 10 microM lactacystin, and caspases were inhibited by 1 microM Z-DEVD-FMK or 20 microM Z-VAD-FMK.. Staurosporin treatment caused a dose- and time-dependent increase in the number of apoptotic cells and in caspase-3 activity. Activation of caspase-2, -4, -8, and -9 was also seen. Caspase activity was increased after 3 hours' incubation with 1 microM staurosporin, which is also the time when most cells became annexin-V-positive. Nuclear changes indicative of apoptosis, viewed with both Hoechst and TUNEL staining, appeared after 4 to 6 hours of staurosporin incubation. Incubation of BLECs with lactacystin caused reduction of proteasome activity and increased apoptosis, evidenced in both the TUNEL assay and caspase-3 activation. Preincubation of lens epithelial cells with caspase inhibitors caused complete inhibition of lactacystin- or staurosporin-induced caspase-3 activation (Z-DEVD-FMK/Z-VAD-FMK) and also of caspase-2, -4, -8, and -9 (Z-VAD-FMK), but the reduction in TUNEL-positive cells was only partial. PS translocation and DNA fragmentation after staurosporin treatment occurred despite complete caspase blockade.. Staurosporin-induced apoptosis in BLECs involves activation of several caspases. Inhibition of the proteasome causes caspase-3 activation and apoptosis. Both staurosporin- and lactacystin-induced apoptosis can be executed in a caspase-independent manner. The present data are useful for understanding of proteolytic mechanisms during apoptosis in lens epithelial cells, which may be an important event in normal lens development as well as in some types of cataract. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; Benzimidazoles; Caspase Inhibitors; Caspases; Cattle; Cells, Cultured; Cysteine Endopeptidases; Dose-Response Relationship, Drug; Enzyme Inhibitors; Epithelial Cells; Fluorescent Dyes; In Situ Nick-End Labeling; Lens, Crystalline; Multienzyme Complexes; Oligopeptides; Proteasome Endopeptidase Complex; Staurosporine; Time Factors | 2000 |
Posttranslational regulation of the retinoblastoma gene family member p107 by calpain protease.
The retinoblastoma protein plays a critical role in regulating the G1/S transition. Less is known about the function and regulation of the homologous pocket protein p107. Here we present evidence for the posttranslational regulation of p107 by the Ca2+-activated protease calpain. Three negative growth regulators, the HMG-CoA reductase inhibitor lovastatin, the antimetabolite 5-fluorouracil, and the cyclic nucleotide dibutyryl cAMP were found to induce cell type-specific loss of p107 protein which was reversible by the calpain inhibitor leucyl-leucyl-norleucinal but not by the serine protease inhibitor phenylmethylsulfonylfluoride, caspase inhibitors, or lactacystin, a specific inhibitor of the 26S proteasome. Purified calpain induced Ca2+-dependent p107 degradation in cell lysates. Transient expression of the specific calpain inhibitor calpastatin blocked the loss of p107 protein in lovastatin-treated cells, and the half-life of p107 was markedly lengthened in lovastatian-treated cells stably transfected with a calpastatin expression vector versus cells transfected with vector alone. The data presented here demonstrate down-regulation of p107 protein in response to various antiproliferative signals, and implicate calpain in p107 posttranslational regulation. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Bucladesine; Calpain; Cyclin B; Cyclin B1; Cysteine Proteinase Inhibitors; Dipeptides; Fluorouracil; Humans; Hydroxymethylglutaryl-CoA Reductase Inhibitors; Ketones; Leupeptins; Lovastatin; Nuclear Proteins; Protein Processing, Post-Translational; Retinoblastoma Protein; Retinoblastoma-Like Protein p107; Tumor Cells, Cultured | 1999 |
Phosphorylation and proteasome-dependent degradation of Bcl-2 in mitotic-arrested cells after microtubule damage.
Treatment of NIH-OVCAR-3 cells with paclitaxel, a microtubule-stabilizing agent, induces mitotic arrest and apoptosis, but also Bcl-2 phosphorylation. We report here that Bcl-2 phosphorylation precedes Bcl-2 down-regulation and that both events are closely associated with mitotic arrest, but are not sufficient for paclitaxel to trigger apoptosis. Indeed, when paclitaxel-treated cells were induced to exit mitosis in the presence of 2-aminopurine, Bcl-2 phosphorylation and Bcl-2 down-regulation were both inhibited. In contrast, when apoptosis was inhibited by a caspase inhibitor or Bcl-2 over-expression, Bcl-2 phosphorylation and down-regulation still occurred. Furthermore, we show that Bcl-2 is degraded in mitosis by the proteasome-dependent pathway since Bcl-2 down-regulation is inhibited by proteasome inhibitors such as MG132, Lactacystin and LLnL. Taken together these results indicate that mitotic spindle damage results in post-translational modifications of Bcl-2 by phosphorylation and degradation. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Apoptosis; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Female; Gene Expression Regulation, Neoplastic; Genes, bcl-2; Humans; Leupeptins; Microtubules; Mitosis; Multienzyme Complexes; Ovarian Neoplasms; Paclitaxel; Phosphorylation; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins c-bcl-2; Tumor Cells, Cultured; Virulence Factors, Bordetella | 1999 |
Bax and Bak proteins require caspase activity to trigger apoptosis in sympathetic neurons.
We show that the pro-apoptotic proteins Bax and Bak trigger apoptosis when over-expressed in sympathetic neurons cultured in the presence of NGF. This effect can be blocked with z-VAD-fmk, a peptide inhibitor of caspases, but not with anti-apoptotic chemical compounds such as antioxidants or proteasome inhibitors. These results demonstrate that in sympathetic neurons Bax and Bak are sufficient to induce apoptosis in the absence of any other apparent cell death stimulus and that their effect is mediated by caspases but does not require reactive oxygen species nor activity of the proteasome. Topics: Acetylcysteine; Amino Acid Chloromethyl Ketones; Animals; Apoptosis; bcl-2 Homologous Antagonist-Killer Protein; bcl-2-Associated X Protein; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Deferoxamine; Membrane Proteins; Nerve Tissue Proteins; Neurons; Phosphodiesterase Inhibitors; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Rats; Superior Cervical Ganglion | 1998 |