leupeptins and Neuroblastoma

Human tetratricopeptide repeat domain 3 (TTC3) is a gene on 21q22.2 within the Down syndrome critical region (DSCR). Earlier studies suggest that TTC3 may be an important regulator in individual development, especially in neural development. As an E3 ligase, TTC3 binds to phosphorylated Akt and silence its activity via proteasomal cascade. Several groups also reported the involvement of TTC3 in familial Alzheimer's disease recently. In addition, our previous work shows that TTC3 also regulates the degradation of DNA polymerase gamma and over-expressed TTC3 protein tends to form insoluble aggregates in cells. In this study, we focus on the solubility and intracellular localization of TTC3 protein. Over-expressed TTC3 tends to form insoluble aggregates over time. The proteasome inhibitor MG132 treatment resulted in more TTC3 aggregates in a short period of time. We fused the fluorescent protein to either terminus of the TTC3 protein and found that the intracellular localization of fluorescent signals are different between the N-terminal tagged and C-terminal tagged proteins. Western blotting revealed that the TTC3 protein is cleaved into fragments of different sizes at multiple sites. The N-terminal sub-fragments of TTC3 are prone to from nuclear aggregates and the TTC3 nuclear import is mediated by signals within the N-terminal 1 to 650 residues. Moreover, over-expressed TTC3 induced a considerable degree of cytotoxicity, and its N-terminal sub-fragments are more potent inhibitors of cell proliferation than full-length protein. Considering the prevalent proteostasis dysregulation in neurodegenerative diseases, these findings may relate to the pathology of such diseases.

Topics: Animals; Bacterial Proteins; Cell Division; Cell Line, Tumor; Cell Nucleus; Chromosomes, Human, Pair 21; Down Syndrome; Gene Expression Regulation; HEK293 Cells; Humans; Leupeptins; Luminescent Proteins; Mice; Nerve Tissue Proteins; Neuroblastoma; Proteasome Endopeptidase Complex; Protein Aggregates; Protein Aggregation, Pathological; Recombinant Fusion Proteins; Red Fluorescent Protein; Transfection; Ubiquitin-Protein Ligases

Neuroblastoma is the most common extracranial solid neuroendocrine cancer and is one of the leading causes of death in children. To improve clinical outcomes and prognosis, discovering new promising drugs and targeted medicine is essential. We found that applying Suberoylanilide hydroxamic acid (SAHA; Vorinostat, a histone deacetylase inhibitor) and MG132 (a proteasome inhibitor) to SH-SY5Y cells synergistically suppressed proliferation, glucose metabolism, migration, and invasion and induced apoptosis and cell cycle arrest. These effects occurred both concentration and time dependently and were associated with the effects observed with inhibitor of growth 5 (ING5) overexpression. SAHA and MG132 treatment increased the expression levels of ING5, PTEN, p53, Caspase-3, Bax, p21, and p27 but decreased the expression levels of 14-3-3, MMP-2, MMP-9, ADFP, Nanog, c-myc, CyclinD1, CyclinB1, and Cdc25c concentration dependently, similar to ING5. SAHA may downregulate miR-543 and miR-196-b expression to enhance the translation of ING5 protein, which promotes acetylation of histones H3 and H4. All three proteins (ING5 and acetylated histones H3 and H4) were recruited to the promoters of c-myc, Nanog, CyclinD1, p21, and p27 for complex formation, thereby regulating the mRNA expression of downstream genes. ING5 overexpression and SAHA and/or MG132 administration inhibited tumor growth in SH-SY5Y cells by suppressing proliferation and inducing apoptosis. The expression of acetylated histones H3 and ING5 may be closely linked to the tumor size of neuroblastomas. In summary, SAHA and/or MG132 can synergistically suppress the malignant phenotypes of neuroblastoma cells through the miRNA-ING5-histone acetylation axis and via proteasomal degradation, respectively. Therefore, the two drugs may serve as potential treatments for neuroblastoma.

Topics: Acetylation; Animals; Antineoplastic Agents; Apoptosis; Biomarkers; Cell Cycle; Cell Line, Tumor; Disease Models, Animal; Energy Metabolism; Gene Expression Regulation, Neoplastic; Histone Deacetylase Inhibitors; Histones; Humans; Immunohistochemistry; Leupeptins; Mice; MicroRNAs; Models, Biological; Neuroblastoma; Signal Transduction; Transcription Factors; Tumor Suppressor Proteins; Vorinostat; Xenograft Model Antitumor Assays

Gene multiplications or point mutations in alpha (α)-synuclein are associated with familial and sporadic Parkinson's disease (PD). An increase in copper (Cu) levels has been reported in the cerebrospinal fluid and blood of PD patients, while occupational exposure to Cu has been suggested to augment the risk to develop PD. We aimed to elucidate the mechanisms by which α-synuclein and Cu regulate dopaminergic cell death. Short-term overexpression of wild type (WT) or mutant A53T α-synuclein had no toxic effect in human dopaminergic cells and primary midbrain cultures, but it exerted a synergistic effect on Cu-induced cell death. Cell death induced by Cu was potentiated by overexpression of the Cu transporter protein 1 (Ctr1) and depletion of intracellular glutathione (GSH) indicating that the toxic effects of Cu are linked to alterations in its intracellular homeostasis. Using the redox sensor roGFP, we demonstrated that Cu-induced oxidative stress was primarily localized in the cytosol and not in the mitochondria. However, α-synuclein overexpression had no effect on Cu-induced oxidative stress. WT or A53T α-synuclein overexpression exacerbated Cu toxicity in dopaminergic and yeast cells in the absence of α-synuclein aggregation. Cu increased autophagic flux and protein ubiquitination. Impairment of autophagy by overexpression of a dominant negative Atg5 form or inhibition of the ubiquitin/proteasome system (UPS) with MG132 enhanced Cu-induced cell death. However, only inhibition of the UPS stimulated the synergistic toxic effects of Cu and α-synuclein overexpression. Our results demonstrate that α-synuclein stimulates Cu toxicity in dopaminergic cells independent from its aggregation via modulation of protein degradation pathways.

Topics: alpha-Synuclein; Animals; Apoptosis; Caspases; Cells, Cultured; Copper; Cysteine Proteinase Inhibitors; Dopaminergic Neurons; Embryo, Mammalian; Gene Expression Regulation; Humans; Leupeptins; Mesencephalon; Mutation; Neuroblastoma; Proteolysis; Rats; Rats, Sprague-Dawley; Signal Transduction; Time Factors; Tyrosine 3-Monooxygenase

Amyotrophic lateral sclerosis (ALS) is a fatal, adult-onset, and progressive neurodegenerative disorder with no cure. Cu/Zn-superoxide dismutase (SOD1) was the first identified protein associated with familial ALS; and aggresome formation of misfolded SOD1 is closely associated with ALS pathogenesis. HDAC6, one of the histone deacetylase family members, has already been demonstrated to play an important role in regulating aggresome formation of misfolded proteins and protecting cells against the toxicity induced by misfolded proteins. In this study, we found that in a cellular model with impaired proteasome activity, the TAR DNA-binding protein 43, which is closely linked with ALS and associated with various neurodegenerative disorders such as frontotemporal lobar degeneration, Alzheimer’s disease, and Parkinson’s disease, can regulate mutant SOD1 aggresome formation through an HDAC6-dependent manner. TDP-43 deficiency did not affect poly-ubiquitination of mutant SOD1, whereas it greatly decreased the expression level of HDAC6, which is required for aggresome formation of ALS-linked mutant SOD1. Moreover, overexpression of siRNA-resistant HDAC6 restored mutant SOD1 aggresome formation in TDP-43-knockdown cells. Thus, our data provide evidence that TDP-43 plays an important role in mutant SOD1 aggresome formation through its regulation of HDAC6.

Topics: Analysis of Variance; Animals; Cell Line, Tumor; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Gene Expression Regulation; Green Fluorescent Proteins; HEK293 Cells; Histone Deacetylase 6; Histone Deacetylases; Humans; Immunoprecipitation; Leupeptins; Mice; Mutation; Neuroblastoma; Protein Aggregates; RNA Interference; RNA, Messenger; Superoxide Dismutase; Superoxide Dismutase-1; Transfection; Tubulin

Recently, cation transport regulator homolog 1 (Chac1) has been identified as a novel pro-apoptotic factor in cells under endoplasmic reticulum (ER) stress. Of the three major ER stress sensors, it is suggested that ATF4 participates in the transcriptional regulation of Chac1 gene expression. The precise characterization of the Chac1 promoter, however, has not yet been elucidated. In this study, we detected the induction of Chac1 mRNA expression using DNA array analysis and RT-PCR of thapsigargin (Tg)-inducible genes in Neuro2a cells. Chac1 mRNA expression was also induced immediately following treatment with tunicamycin (Tm) and brefeldin A. Characterization of the mouse Chac1 promoter activity using a luciferase reporter assay revealed that the CREB/ATF element and amino acid response element in the mouse Chac1 promoter are functional and respond to Tm stimulation and ATF4 overexpression. Mutations in either element in the Chac1 promoter did not inhibit the responsiveness of this promoter to Tm and ATF4; however, mutations in both of these elements dramatically decreased the basal activity and response to ER stress stimuli. In addition to the transcriptional regulation, we found that Chac1 protein expression was only detected in the presence of MG132, a proteasome inhibitor, even though mouse Chac1 gene was transiently overexpressed in Neuro2a cells. Taken together, we are the first to demonstrate the transcriptional and post-translational regulation of Chac1 expression in a neuronal cell line.

Topics: Activating Transcription Factor 4; Animals; Base Sequence; Blotting, Western; Cell Line, Tumor; Connexins; Cysteine Proteinase Inhibitors; Endoplasmic Reticulum Stress; gamma-Glutamylcyclotransferase; Gene Expression Regulation; Intracellular Signaling Peptides and Proteins; Leupeptins; Mice; Molecular Sequence Data; Mutation; Neuroblastoma; Promoter Regions, Genetic; Protein Biosynthesis; Reverse Transcriptase Polymerase Chain Reaction; Thapsigargin; Transcription, Genetic; Tunicamycin

Neuroblastma cell lines contain a side-population of cells which express stemness markers. These stem-like cells may represent the potential underlying mechanism for resistance to conventional therapy and recurrence of neuroblastoma in patients.. To develop novel strategies for targeting the side-population of neurobastomas, we analyzed the effects of 13-cis-retinoic acid (RA) combined with the proteasome inhibitor MG132. The short-term action of the treatment was compared with effects after a 5-day recovery period during which both chemicals were withdrawn. RA induced growth arrest and differentiation of SH-SY5Y and SK-N-BE(2) neuroblastoma cell lines. Inhibition of the proteasome caused apoptosis in both cell lines, thus, revealing the critical role of this pathway in the regulated degradation of proteins involved in neuroblastoma proliferation and survival. The combination of RA with MG132 induced apoptosis in a dose-dependent manner, in addition to promoting G2/M arrest in treated cultures. Interestingly, expression of stem cell markers such as Nestin, Sox2, and Oct4 were reduced after the recovery period of combined treatment as compared with untreated cells or treated cells with either compound alone. Consistent with this, neurosphere formation was significantly impaired by the combined treatment of RA and MG132.. Given that stem-like cells are associated with resistant to conventional therapy and are thought to be responsible for relapse, our results suggest that dual therapy of RA and proteasome inhibitor might be beneficial for targeting the side-population of cells associated residual disease in high-risk neuroblastoma.

Topics: Apoptosis; Blotting, Western; Cell Differentiation; Cell Line, Tumor; Cell Proliferation; Dose-Response Relationship, Drug; Drug Synergism; Flow Cytometry; G2 Phase Cell Cycle Checkpoints; Humans; Leupeptins; Microscopy, Confocal; Neoplastic Stem Cells; Nestin; Neuroblastoma; Octamer Transcription Factor-3; Proteasome Inhibitors; Side-Population Cells; SOXB1 Transcription Factors; Time Factors; Tretinoin

Mutations in the parkin gene underlie a familial form of Parkinson's disease known as autosomal recessive juvenile Parkinsonism (AR-JP). Dysfunction of parkin, a ubiquitin E3 ligase, has been implicated in the accumulation of ubiquitin proteasome system-destined substrates and eventually leads to cell death. However, regulation of parkin enzymatic activity is incompletely understood. Here we investigated whether the ubiquitin E3 ligase activity of parkin could be regulated by neddylation. We found that parkin could be a target of covalent modification with NEDD8, a ubiquitin-like posttranslational modifier. In addition, NEDD8 attachment caused an increase of parkin activity through the increased binding affinity for ubiquitin-conjugating E2 enzyme as well as the enhanced formation of the complex containing parkin and substrates. These findings point to the functional importance of NEDD8 and suggest that neddylation is one to the diverse modes of parkin regulation, potentially linking it to the pathogenesis of AR-JP.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Animals; Cells, Cultured; Cycloheximide; Embryo, Mammalian; Gene Expression Regulation; Hippocampus; Humans; Immunoprecipitation; Leupeptins; Mutation; NEDD8 Protein; Neuroblastoma; Neurotoxins; Protein Binding; Protein Synthesis Inhibitors; Rats; RNA, Small Interfering; Sincalide; Stem Cells; Subcellular Fractions; Time Factors; Transfection; Ubiquitin-Protein Ligases; Ubiquitination; Ubiquitins

Sodium-coupled, high-affinity choline transporters (CHTs) are inhibited by 3-morpholinosydnonimine (SIN-1) [peroxynitrite (ONOO⁻) donor]; ONOO⁻ can be produced from nitric oxide and reactive oxygen species during neurodegeneration. SIN-1 rapidly increases CHT internalization from the cell surface, and this correlates with decreased choline uptake. This study addresses mechanisms by which SIN-1 inhibits CHT function in human neuronal SH-SY5Y cells. Thus, mutant L531A-CHT, which does not constitutively internalize into cells by a clathrin-mediated process, is resistant to SIN-1 effects. This suggests that CHT inhibition is not due to oxidative-nitrosative inactivation of the protein and that decreased levels of cell surface CHT in SIN-1-treated cells is related to alterations in its trafficking and subcellular disposition. Dominant-negative proteins AP180C and dynamin-K44A, which interfere with clathrin-mediated and dynamin-dependent endocytosis, respectively, attenuate CHT inhibition by SIN-1. CHT in both vehicle- and SIN-1-treated cells colocalizes with Rab7, Rab9, and Lamp-1 in late endosomes and lysosomes to a similar extent. Lysosome inhibitors increase choline uptake, suggesting that CHT proteins are normally degraded by lysosomes, and this is not altered by oxidative stress. Unexpectedly, inhibitors of proteasomes, but not lysosomes, attenuate SIN-1-mediated inhibition of choline uptake, indicating that proteasomal degradation plays a role in regulating CHT disposition in SIN-1-treated cells. SIN-1 treatment also enhances CHT ubiquitination. Thus, CHT inhibition in SIN-1-treated cells is mediated by proteasomal degradation, which differs from inhibitory mechanisms for some neurotransmitter transporters under similar conditions. Increased oxidative-nitrosative stress in the microenvironment of cholinergic nerve terminals would diminish cholinergic transmission by reducing choline availability for ACh synthesis.

Topics: Cell Line, Transformed; Cell Line, Tumor; Choline; Clathrin; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Endocytosis; Endosomes; Hemicholinium 3; Humans; Leupeptins; Luminescent Proteins; Lysosomes; Membrane Transport Proteins; Molsidomine; Mutation; Neuroblastoma; Nitric Oxide; Nitric Oxide Donors; Peroxynitrous Acid; Protein Transport; rab5 GTP-Binding Proteins; Time Factors; Transfection; Tritium; Ubiquitination

Nitric oxide is linked with neurodegeneration in Parkinson's disease (PD) through the involvement of both inducible (iNOS) and neuronal nitric oxide synthase (nNOS). While non-selective NOS inhibitors are neuroprotective, the role of nNOS has not been determined using selective NOS inhibitors. The present study investigated the neuroprotective effect of selective iNOS and nNOS inhibitors on MPP(+)- and MG-132-induced cell death in cell lines with differing levels of nNOS expression. Inhibition of endogenously expressed nNOS by 7-NI and ARR17477 enhanced the toxicity of MPP(+) and MG-132 in N1E-115 cells, whereas in transfected SH-SY5Y cells overexpressing nNOS, ARR17477 and 7-NI protected against MPP(+)- and MG-132-induced cell death. In contrast, inhibition of iNOS by 1400W was ineffective in preventing MPP(+) and MG-132 toxicity in these cell lines. These results suggest a dual role for NOS in dopaminergic cell viability. nNOS is protective against toxic insult when produced endogenously. When nNOS is overexpressed, it becomes neurotoxic to cells suggesting that inhibition of nNOS may be a promising strategy to prevent cell death in PD.

Topics: 1-Methyl-4-phenyl-1,2,3,6-tetrahydropyridine; Amidines; Animals; Asparagine; Caspase 3; Cell Death; Cell Line, Tumor; Citrulline; Dopamine; Dopamine Plasma Membrane Transport Proteins; Dose-Response Relationship, Drug; Enzyme Inhibitors; Humans; Indazoles; L-Lactate Dehydrogenase; Leupeptins; Mice; Neuroblastoma; Neurotoxins; Nitric Oxide Synthase Type I; Thiophenes; Transfection; Tritium; Tyrosine 3-Monooxygenase; Vesicular Monoamine Transport Proteins

Abnormal iron accumulation is linked to a variety of neurological disorders and may contribute to the progressive damage seen in these diseases. The biochemical processes responsible for iron accumulation are not known but are likely to entail alteration in transport into injured brain areas. The major transport protein responsible for uptake of iron is divalent metal transporter 1 (DMT1) and recent studies demonstrate that the 1B species is regulated post-translationally by degradation via the proteasomal pathway. As reported in this paper, the E3 ligase, parkin, when over-expressed in SH-SY5Y cells, results in a decrease in 1B-DMT1 isoforms and also a significant reduction in manganese transport and toxicity. Incubating cells over-expressing parkin with the proteasomal inhibitor, MG-132, restores 1B-DMT1 levels emphasizing that the observed changes are caused by degradation via the proteasomal pathway. Expression of the 1B species of DMT1 was also shown to be elevated in human lymphocytes containing a homozygous deletion of exon 4 of parkin and in brains of parkin knockout animals. Immunoprecipitation and immunofluorescent studies confirm that parkin co-localizes with DMT1 in SH-SY5Y cells transfected with wild-type parkin. These results demonstrate that parkin is the E3 ligase responsible for ubiquitination of the 1B species of DMT1.

Topics: Adult; Animals; B-Lymphocytes; Cation Transport Proteins; Cell Death; Cells, Cultured; Chlorides; Cysteine Proteinase Inhibitors; Exons; Gene Expression Regulation; Humans; Immunoprecipitation; Leupeptins; Male; Manganese; Manganese Compounds; Mice; Mice, Knockout; Mutation; Neuroblastoma; Parkinson Disease; Proteasome Endopeptidase Complex; Protein Isoforms; Transfection; Ubiquitin-Protein Ligases

Mutations in the gene encoding the E3 ubiquitin-protein ligase parkin have been shown to be a common genetic cause of familial early-onset Parkinson's disease (PD). In addition to its function in the ubiquitin-proteasome system (UPS), parkin has been ascribed general neuroprotective properties. Stress and mutation induced decreases in parkin solubility leading to compromised cytoprotection have recently been reported. We systematically investigated whether PD-related stresses including MG132 and epoxomicin (proteasomal impairment), tunicamycin (unfolded protein stress), and rotenone (mitochondrial dysfunction) resulted in expressional changes of parkin and other E3 ubiquitin ligases (dorfin, SIAH-1). Rotenone and tunicamycin did not change parkin mRNA levels, whereas proteasomal inhibition resulted in a reduction of parkin mRNA in PC12 cells as well as in SH-SY5Y cells. Therefore, surprisingly, cells did not react with a compensatory parkin upregulation under proteasomal inhibition, although, in parallel, parkin protein shifted to the insoluble fraction, reducing soluble parkin levels in the cytosol. Since the mRNA of the parkin-coregulated gene PACRG paralleled the parkin mRNA at least partly, we suspect a promoter-driven mechanism. Our study, therefore, shows a link between proteasomal impairment and parkin expression levels in cell culture, which is intriguing in the context of the described and debated proteasomal dysfunction in the substantia nigra of PD patients.

Topics: Animals; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Enzyme Inhibitors; Gene Expression Regulation; Humans; Leupeptins; Neuroblastoma; Nuclear Proteins; Oligopeptides; PC12 Cells; Proteasome Inhibitors; Rats; RNA, Messenger; Rotenone; Time Factors; Tunicamycin; Ubiquitin-Protein Ligases

Mutations in PARK7 DJ-1 have been associated with autosomal-recessive early-onset Parkinson's disease (PD). This gene encodes for an atypical peroxiredoxin-like peroxidase that may act as a regulator of transcription and a redox-dependent chaperone. Although large gene deletions have been associated with a loss-of-function phenotype, the pathogenic mechanism of several missense mutations is less clear. By performing a yeast two-hybrid screening from a human fetal brain library, we identified TRAF and TNF receptor-associated protein (TTRAP), an ubiquitin-binding domain-containing protein, as a novel DJ-1 interactor, which was able to bind the PD-associated mutations M26I and L166P more strongly than wild type. TTRAP protected neuroblastoma cells from apoptosis induced by proteasome impairment. In these conditions, endogenous TTRAP relocalized to a detergent-insoluble fraction and formed cytoplasmic aggresome-like structures. Interestingly, both DJ-1 mutants blocked the TTRAP protective activity unmasking a c-jun N-terminal kinase (JNK)- and p38-MAPK (mitogen-activated protein kinase)-mediated apoptosis. These results suggest an active role of DJ-1 missense mutants in the control of cell death and position TTRAP as a new player in the arena of neurodegeneration.

Topics: Antineoplastic Agents; Apoptosis; Brain Neoplasms; Cell Line; DNA-Binding Proteins; Dopamine; Enzyme Activation; Humans; Inclusion Bodies; Intracellular Signaling Peptides and Proteins; JNK Mitogen-Activated Protein Kinases; Leupeptins; Mutation, Missense; Neuroblastoma; Nuclear Proteins; Oncogene Proteins; Oxidative Stress; p38 Mitogen-Activated Protein Kinases; Parkinson Disease; Phosphoric Diester Hydrolases; Protein Binding; Protein Deglycase DJ-1; Substantia Nigra; Transcription Factors; Two-Hybrid System Techniques

The 26S proteasome plays a major role in degradation of abnormal proteins within the cell. The indirect antioxidant including sulforaphane (SFN) protects cells from oxidative damage by increasing the expression of Nrf2-target genes. It has been observed that the expression of multiple subunits of the proteasome was up-regulated by indirect antioxidants through the Nrf2 pathway. In the current study, the role of SFN in amyloid beta(1-42) (Abeta(1-42))-induced cytotoxicity has been investigated in murine neuroblastoma cells. Treatment with SFN protected cells from Abeta(1-42)-mediated cell death in Neuro2A and N1E 115 cells. Inhibition of proteasome activities by MG132 could abolish the protective effect of SFN against Abeta(1-42). Neuro2A cells, which were stably overexpressing the catalytic subunit of the proteasome PSMB5, showed an elevated resistance toward Abeta(1-42) toxicity compared to control cells. Furthermore, the in vitro assay demonstrated that the Abeta(1-42) peptide is degraded by the proteasome fraction. These results suggest that proteasome-inducing indirect antioxidants may facilitate the removal of the Abeta(1-42) peptide and lead to the amelioration of abnormal protein-associated etiologies.

Topics: Amyloid beta-Peptides; Animals; Antioxidants; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Cytoprotection; Dose-Response Relationship, Drug; Isothiocyanates; Leupeptins; Mice; Neuroblastoma; Neurons; Peptide Fragments; Promoter Regions, Genetic; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Sulfoxides; Thiocyanates; Transfection; Up-Regulation

Dopamine at 100-500 microM has toxic effects on human SH-SY5Y neuroblastoma cells, manifested as apoptotic cell loss and strong autophagy. The molecular mechanisms and types of dopamine-induced cell death are not yet well known. Their identification is important in the study of neurodegenerative diseases that specifically involve dopaminergic neurons. We looked for changes in expression and content of proteins involved in apoptosis and autophagy after dopamine treatment. All the changes found were prevented by avoiding dopamine oxidation with N-acetylcysteine, indicating a key role for the products of dopamine oxidation in dopamine toxicity. As early as 1-2h after treatment we found an increase in hypoxia-inducible factor-1alpha (HIF-1alpha) and an accumulation of ubiquitinated proteins. Proteins regulated by HIF-1alpha and involved in apoptosis and/or autophagy, such as p53, Puma and Bnip3, were subsequently increased. However, apoptotic parameters (caspase-3, caspase-7, PARP) were only activated after 12h of 500muM dopamine treatment. Autophagy, monitored by the LC3-II increase after LC3-I linkage to autophagic vacuoles, was evident after 6h of treatment with both 100 and 500 microM dopamine. The mTOR pathway was inhibited by dopamine, probably due to the intracellular redox changes and energy depletion leading to AMPK activation. However, this mechanism is not sufficient to explain the high LC3-II activation caused by dopamine: the LC3-II increase was not reversed by IGF-1, which prevented this effect when caused by the mTOR inhibitor rapamycin. Our results suggest that the aggregation of ubiquitinated non-degraded proteins may be the main cause of LC3-II activation and autophagy. As we have reported previously, cytosolic dopamine may cause damage by autophagy in neuroblastoma cells (and presumably in dopaminergic neurons), which develops to apoptosis and leads to cell degeneration.

Topics: Analysis of Variance; Apoptosis Regulatory Proteins; Autophagy; Caspase 3; Cell Line, Tumor; Cell Survival; Cysteine Proteinase Inhibitors; Dopamine; Dopamine Agents; Dose-Response Relationship, Drug; Fluoresceins; Humans; Hypoxia-Inducible Factor 1, alpha Subunit; Leupeptins; Microscopy, Electron, Transmission; Microtubule-Associated Proteins; Nerve Tissue Proteins; Neuroblastoma; Oncogene Protein v-akt; Oxidative Stress; Phosphatidylinositol 3-Kinases; Protein Kinases; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Signal Transduction; TOR Serine-Threonine Kinases

The transcription factor p73, a member of the p53 family, mediates cell-cycle arrest and apoptosis in response to DNA damage-induced cellular stress, acting thus as a proapoptotic gene. Similar to p53, p73 activity is regulated by post-translational modification, including phosphorylation, acetylation and ubiquitylation. In C. elegans, the F-box protein FSN-1 controls germline apoptosis by regulating CEP-1, the single ancestral p53 family member. Here we report that FBXO45, the human ortholog of FSN-1, binds specifically to p73 triggering its proteasome-dependent degradation. Importantly, SCF(FBXO45) ubiquitylates p73 both in vivo and in vitro. Moreover, siRNA-mediated depletion of FBXO45 stabilizes p73 and concomitantly induces cell death in a p53-independent manner. All together, these results show that the orphan F-box protein FBXO45 regulates the stability of p73, highlighting a conserved pathway evolved from nematode to human by which the p53 members are regulated by an SCF-dependent mechanism.

Topics: Animals; Breast Neoplasms; Cell Death; Cell Line; Cell Line, Transformed; Cell Line, Tumor; CHO Cells; Cricetinae; Cricetulus; DNA-Binding Proteins; F-Box Proteins; Green Fluorescent Proteins; HeLa Cells; Hemagglutinins; Humans; Kidney; Leupeptins; Mutation; Neuroblastoma; Nuclear Proteins; Proteasome Endopeptidase Complex; Proteasome Inhibitors; Protein Binding; RNA, Small Interfering; Substrate Specificity; Temperature; Transfection; Tumor Protein p73; Tumor Suppressor Proteins; Ubiquitin-Protein Ligases; Ubiquitination

Prion diseases are transmissible neurodegenerative diseases caused by a conformational isoform of the prion protein (PrP), a host-encoded cell surface sialoglycoprotein. Recent evidence suggests a cytosolic fraction of PrP (cyPrP) functions either as an initiating factor or toxic element of prion disease. When expressed in cultured cells, cyPrP acquires properties of the infectious conformation of PrP (PrP(Sc)), including insolubility, protease resistance, aggregation, and toxicity. Transgenic mice (2D1 and 1D4 lines) that coexpress cyPrP and PrP(C) exhibit focal cerebellar atrophy, scratching behavior, and gait abnormalities suggestive of prion disease, although they lack protease-resistant PrP. To determine if the coexpression of PrP(C) is necessary or inhibitory to the phenotype of these mice, we crossed Tg1D4(Prnp(+/+)) mice with PrP-ablated mice (TgPrnp(o/o)) to generate Tg1D4(Prnp(o/o)) mice and followed the development of disease and pathological phenotype. We found no difference in the onset of symptoms or the clinical or pathological phenotype of disease between Tg1D4(Prnp(+/+)) and Tg1D4(Prnp(o/o)) mice, suggesting that cyPrP and PrP(C) function independently in the disease state. Additionally, Tg1D4(Prnp(o/o)) mice were resistant to challenge with mouse-adapted scrapie (RML), suggesting cyPrP is inaccessible to PrP(Sc). We conclude that disease phenotype and cellular toxicity associated with the expression of cyPrP are independent of PrP(C) and the generation of typical prion disease.

Topics: Animals; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Cytosol; Disease Progression; Gene Expression; Injections, Intraventricular; Leupeptins; Mice; Mice, Inbred C57BL; Mice, Transgenic; Neuroblastoma; Prions; PrPSc Proteins; Transfection

p120-catenin contributes to the cadherin-mediated adhesion and aggregation of cells. mu-Calpain was activated and p120-catenin was degraded after 36 h of ischemia in differentiated SH-SY5Y cells. Calpain inhibitors Cbz-Val-Phe-H (MDL28170, 20 microM) and N-acetyl-leucyl-leucyl-norleucinal (ALLN, 20 microM) increased the levels of dephosphorylated p120-catenin, aggregation, and cell survival as detected by reduced LDH release in ischemic cells. However, a proteasome inhibitor lactacystin had no such effects. This is the first report of the calpain-mediated degradation of p120-catenin and an association between the level of dephosphorylated p120-catenin and cell aggregation in ischemic neuronal cells.

Topics: Acetylcysteine; Calpain; Catenins; Cell Adhesion Molecules; Cell Aggregation; Cell Death; Cell Line, Tumor; Delta Catenin; Dipeptides; Humans; Ischemia; Leupeptins; Neuroblastoma; Phosphoproteins

Mutations in parkin and alpha-synuclein (alpha-syn) are linked to heritable forms of Parkinson's disease (PD). Recently, it has been shown that parkin mitigates alpha-syn-induced neuronal cell death in animal and tissue culture models, suggesting that there is a functional relationship between these two proteins. Although the mechanism by which parkin protects cells from alpha-syn-induced cytotoxicity remains elusive, it is tempting to speculate that parkin might directly regulate the normal metabolism and aggregation of alpha-syn. In the current study, we show that neither the suppression of endogenous parkin expression nor ectopic overexpression affects the steady-state levels of endogenous alpha-syn expression, overall aggregation of this protein, or breakdown of pre-formed aggregates in human neuroblastoma cells. These results suggest that parkin is not directly involved in the metabolism of alpha-syn, its aggregation, or the clearance of pre-formed aggregates.

Topics: alpha-Synuclein; Analysis of Variance; Blotting, Western; Cell Line, Tumor; Cysteine Proteinase Inhibitors; Gene Expression; Humans; Leupeptins; Neuroblastoma; RNA, Small Interfering; Transfection; Ubiquitin-Protein Ligases

Microtubule-stabilizing and -destabilizing proteins play a crucial role in regulating the dynamic instability of microtubules during neuronal development and synaptic transmission. The microtubule-destabilizing protein SCG10 is a neuron-specific protein implicated in neurite outgrowth. The SCG10 protein is significantly reduced in mature neurons, suggesting that its expression is developmentally regulated. In contrast, the microtubule-stabilizing protein tau is expressed in mature neurons and its function is essential for the maintenance of neuronal polarity and neuronal survival. Thus, the establishment and maintenance of neuronal polarity may down-regulate the protein level/function of SCG10. In this report, we show that treatment of PC12 cells and neuroblastoma cells with the microtubule-stabilizing drug Taxol induced a rapid degradation of the SCG10 protein. Consistently, overexpression of tau protein in neuroblastoma cells also induced a reduction in SCG10 protein levels. Calpain inhibitor MDL-28170, but not caspase inhibitors, blocked a significant decrease in SCG10 protein levels. Collectively, these results indicate that tau overexpression and Taxol treatment induced a calpain-dependent degradation of the microtubule-destabilizing protein SCG10. The results provide evidence for the existence of an intracellular mechanism involved in the regulation of SCG10 upon microtubule stabilization.

Topics: Animals; Blotting, Western; Calpain; Cell Line; Cysteine Proteinase Inhibitors; Dose-Response Relationship, Drug; Drug Interactions; Enzyme Activation; Gene Expression Regulation; Humans; Leupeptins; Membrane Proteins; Neuroblastoma; Paclitaxel; Rats; Stathmin; tau Proteins; Transfection; Tubulin Modulators

Transcription factor GATA-2 is expressed in a number of tissues, including hematopoietic stem and progenitor cells, and is crucial for the proliferation and survival of hematopoietic cells. To further characterize the function of GATA-2, we examined the cellular turnover mechanism of GATA-2. In P815 cells, the half-life of endogenous GATA-2 was found to be as short as 30 min after cycloheximide treatment. This short half-life was reproducible in other hematopoietic and neuroblastoma cell lines with moderate variation. We also found that ultraviolet (UV)-C irradiation markedly represses the GATA-2 protein level by facilitating the degradation process. Since treatment of the cells with the proteasome inhibitor MG132 or clasto-Lactacystin substantially abrogated the effects of cycloheximide and UV-C irradiation and increased the expression level of both endogenous and transfected GATA-2, the degradation of GATA-2 seems to occur through the proteasome pathway. Structure-function analyses with the GAL4-DNA binding domain (GBD)-GATA-2 fusion protein and GATA-2 deletion mutants suggested that the protein degradation regulatory elements of GATA-2 reside in three regions, two of which overlap with the transactivation domain. We also detected poly ubiquitinated forms of GATA-2. Taken together, these results demonstrate that GATA-2 is turned over rapidly through the ubiquitin-proteasome pathway.

Topics: Acetylcysteine; Animals; Cells, Cultured; Cycloheximide; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; GATA2 Transcription Factor; Gene Deletion; Gene Expression Regulation; Half-Life; Humans; Leukemia, Experimental; Leupeptins; Mice; Mutation; Neuroblastoma; Proteasome Endopeptidase Complex; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Transcription Factors; Transcriptional Activation; Ubiquitins; Ultraviolet Rays

Decreased proteasome activity is an important pathology in Parkinson's disease (PD), which is related to cell death and Lewy body formation. In this study, we show that p53-activity may correlate with neuronal death via the mitochondrial pathway in PD model. The proteasome inhibitor, MG132, induced the accumulation of p53 in human dopaminergic neuroblastoma SH-SY5Y cells. The increased stabilization of p53 upregulated the level of Bax and mitochondrial depolarization. These events were inhibited by the p53 inhibitor, pifithrin-alpha (PFT). Cell viability analyzes demonstrated that PFT partially prevented MG132-induced cell death. These results suggest that p53 is a candidate as an intermediary between the proteasome system and mitochondria-related neuronal death in PD.

Topics: bcl-2-Associated X Protein; Benzimidazoles; Benzothiazoles; Blotting, Western; Carbocyanines; Cell Death; Cell Line, Tumor; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dopamine; Dose-Response Relationship, Drug; Drug Interactions; Fluorescent Antibody Technique; Humans; Leupeptins; Membrane Potentials; Mitochondria; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Proto-Oncogene Proteins; Proto-Oncogene Proteins c-bcl-2; Thiazoles; Time Factors; Toluene; Tumor Suppressor Protein p53

Inhibition of proteasome activity is associated with a reduction in proliferation and apoptosis in cancer cells, depending upon the extent of inhibition. We have reported that a minimal inhibition of proteasome activity prevented adenosine 3'5'-cyclic monophosphate (cAMP)-induced differentiation and caused apoptosis in murine neuroblastoma (NB) cells in culture. In order to establish whether an elevated cAMP level increases the sensitivity of proteasome to its inhibitors, MG-132 and lactacystin (proteasome inhibitors) were added concomitantly with a stimulator of adenylate cyclase (prostaglandin A1) and an inhibitor of cyclic nucleotide phosphodiesterase (RO20-1724). Results showed that concentrations of MG-132 that did not reduce or that minimally inhibited proteasome activity also did not affect the proliferation of undifferentiated NB cells. However, these concentrations of MG-132 in the presence of an elevated cAMP level markedly inhibited proteasome activity and caused extensive cell death. Similar results were obtained with lactacystin. In normal murine fibroblasts, cAMP-induced reduction in proliferation was not affected by any concentration of MG-132 used in this study. These results suggest that proteasome exhibits increased sensitivity to its inhibitors following an elevation of cAMP level in NB cells, but not in normal fibroblasts, and that this may account for the enhanced cell death in NB cells. Thus, the strategy of using low doses of a proteasome inhibitor in combination with a cAMP-stimulating agent may be useful in pre-clinical and clinical studies on NB tumor because of the selectivity of the effect on cancer cells.

Topics: 4-(3-Butoxy-4-methoxybenzyl)-2-imidazolidinone; Acetylcysteine; Adenylyl Cyclase Inhibitors; Animals; Cell Differentiation; Cell Survival; Cyclic AMP; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Fibroblasts; Leupeptins; Mice; Multienzyme Complexes; Neuroblastoma; Phosphodiesterase Inhibitors; Proteasome Endopeptidase Complex; Tumor Cells, Cultured

Huntington's disease is a progressive neurodegenerative disorder that is associated with a CAG repeat expansion in the gene encoding huntingtin. We found that a 60-kDa protein was increased in Neuro2a cells expressing the N-terminal portion of huntingtin with expanded polyglutamine. We purified this protein, and, using mass spectrometry, identified it as p62, an ubiquitin-associated domain-containing protein. A specific p62 antibody stained the ubiquitylated polyQ inclusions in expanded polyglutamine-expressing cells, as well as in the brain of the huntingtin exon 1 transgenic mice. Furthermore, the level of p62 protein and mRNA was increased in expanded polyglutamine-expressing cells. We also found that p62 formed aggresome-like inclusions when p62 was increased in normal Neuro2a cells by a proteasome inhibitor. Knock-down of p62 does not affect the formation of aggresomes or polyglutamine inclusions, suggesting that p62 is recruited to the aggresome or inclusions secondary to their formation. These results suggest that p62 may play important roles as a responsive protein to a polyglutamine-induced stress rather than as a cross-linker between ubiquitylated proteins.

Topics: Animals; Antineoplastic Agents; Brain; Carrier Proteins; Cell Count; Cell Fractionation; Cell Line, Tumor; Cell Nucleus; Chromatography, High Pressure Liquid; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Drug Interactions; Exons; Fluorescent Antibody Technique; Gene Expression Regulation; Green Fluorescent Proteins; Huntingtin Protein; Immunoblotting; Immunoprecipitation; Inclusion Bodies; Indoles; Leupeptins; Male; Mass Spectrometry; Mice; Mice, Knockout; Nerve Tissue Proteins; Neuroblastoma; Neurons; Nocodazole; Nuclear Proteins; Peptides; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Messenger; Sequence Analysis, Protein; Time Factors; Transcription Factor TFIIH; Transcription Factors; Transfection; Ubiquitin

To seek a novel therapeutic approach to neuroblastoma (NBL), we used three NBL cell lines (SK-N-DZ, NH12, and SK-N-SH) to examine the underlining molecular mechanisms of cellular reactions and sensitivity to all-trans-retinoic acid (ATRA). SK-N-DZ cells expressed relatively high levels of retinoic acid receptor alpha (RAR-alpha) and underwent ATRA-induced cell death that was blocked by an RAR-alpha antagonist. By contrast, RAR-alpha expression gradually decreased in NH12 and SK-N-SH cells, which did not experience increased cell death in response to ATRA. We report here the ubiquitin-dependent down-regulation of RAR-alpha expression during ATRA treatment. Our data suggest that SK-N-DZ cells have a defect in RAR-alpha down-regulation, resulting in sustained high expression of RAR-alpha that confers high sensitivity to ATRA. Accordingly, treatment with a proteasome inhibitor dramatically increased ATRA-induced cell death in NH12 and SK-N-SH cell lines. Our results reveal the crucial involvement of the RAR-alpha signaling pathway in NBL cell death and show that three NBL cell lines are differentially sensitive to ATRA. These data suggest a potential novel therapy for NBL involving retinoic acid treatment combined with the inhibition of RAR-alpha degradation.

Topics: Benzoates; Cell Division; Cell Line, Tumor; Chromans; Humans; Leupeptins; Neuroblastoma; Proteasome Inhibitors; Receptors, Retinoic Acid; Retinoic Acid Receptor alpha; RNA, Messenger; Tretinoin; Ubiquitin

Proteasome activity is essential during cAMP-induced terminal differentiation of a murine neuroblastoma cell line (NBP2). However, the mechanisms through which proteasome affects NBP2 differentiation have not been characterized. We hypothesized that proteasome is required to implement the differentiation-mediated effects on cell cycle, and its partial inhibition during differentiation may have adverse consequences. Here we show that partial inhibition of proteasome during cAMP-induced differentiation of NBP2 cells causes apoptosis. Whereas differentiation induced growth arrest at G1 phase, partial proteasome inhibition during differentiation resulted in the accumulation of cells at G2M phase. Cell cycle data correlated with the level of cyclin-dependent kinase inhibitors p21WAF and p27Kip1, and cyclin A. While the level of p21 and p27 increased, the level of cyclin A decreased upon differentiation. In contrast, cells treated with proteasome inhibitor in the presence of cAMP-inducing agents showed increased levels of p21 and cyclin A early in the course of differentiation. However, the level of p21 and p27, but not cyclin A, decreased later during concomitant differentiation and partial proteasome inhibition when cells were undergoing apoptosis. Our data suggest that differentiation-mediated growth arrest is dependent on the temporal activity of cell cycle proteins. Partial inhibition of proteasome interferes with differentiation events partly by stabilizing cell cycle proteins and this triggers apoptosis. Thus, differentiating drugs combined with partial proteasome inhibition may impart higher therapeutic efficacy than differentiating agents alone for the treatment of neuroblastoma tumors.

Topics: Animals; Apoptosis; Caspase 3; Caspase Inhibitors; Caspases; Cell Cycle; Cell Cycle Proteins; Cell Differentiation; Cyclic AMP; Cyclin A; Cyclin-Dependent Kinase Inhibitor p21; Cyclin-Dependent Kinase Inhibitor p27; Cyclins; Cysteine Endopeptidases; Enzyme Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Leupeptins; Mice; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Retroviridae; Transfection; Tumor Cells, Cultured; Tumor Suppressor Proteins

Parkinson's disease is characterized by loss of nigral dopaminergic neurons and the presence of cytoplasmic inclusions known as Lewy bodies. alpha-Synuclein and its interacting partner synphilin-1 are among constituent proteins in these aggregates. The presence of ubiquitin and proteasome subunits in these inclusions supports a role for this protein degradation pathway in the processing of proteins involved in this disease. To begin elucidating the kinetics of synphilin-1 in cells, we studied its degradation pathway in HEK293 cells that had been engineered to stably express FLAG-tagged synphilin-1. Pulse-chase experiments revealed that this protein is relatively stable with a half-life of about 16 h. Treatment with proteasome inhibitors resulted in attenuation of degradation and the accumulation of high molecular weight ubiquitinated synphilin-1 in immunoprecipitation/immunoblot experiments. Additionally, proteasome inhibitors stimulated the formation of peri-nuclear inclusions which were immunoreactive for synphilin-1, ubiquitin and alpha-synuclein. Cell viability studies revealed increased susceptibility of synphilin-1 over-expressing cells to proteasomal dysfunction. These observations indicate that synphilin-1 is ubiquitinated and degraded by the proteasome. Accumulation of ubiquitinated synphilin-1 due to impaired clearance results in its aggregation as peri-nuclear inclusions and in poor cell survival.

Topics: Acetylcysteine; alpha-Synuclein; Animals; Blotting, Western; Carrier Proteins; Cell Line; Cell Survival; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Dimethyl Sulfoxide; Humans; Inclusion Bodies; Intracellular Signaling Peptides and Proteins; Kidney; Leupeptins; Macromolecular Substances; Mice; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Precipitin Tests; Proteasome Endopeptidase Complex; Protein Processing, Post-Translational; Synucleins; Transfection; Ubiquitin

1. The ubiquitin-proteasome pathway is involved in a variety of cellular functions in mammalian cells. The role of proteasome, however, in the course of cell differentiation is not well characterized. We hypothesized that proteasome activity might be essential during neuronal cell differentiation. 2. To investigate the role of proteasome during neuronal differentiation, we made use of a murine neuroblastoma cell line (NBP2) that terminally differentiates into mature neurons upon elevation of the intracellular level of adenosine 3',5'-cyclic monophosphate (cAMP). To monitor proteasome activity in NBP2 cells, we integrated an expression cassette for a short-lived green fluorescent protein (d2EGFP) into these cells, which were designated as NBP2-PN25. When NBP2-PN25 cells were treated with a proteasome inhibitor, lactacystin or MG132, a dose-dependent increase in the constitutive levels of d2EGFP expression was detected. 3. We also found that proteasome inhibition by lactacystin during the cAMP-induced differentiation of NBP2-PN25 cells triggered cell death. Both lactacystin and cAMP induction reduced the expression of mRNA for the differentiation-associated genes, such as N-myc and cyclin B1. While cAMP-inducing agents decreased the level of N-myc and cyclin B1 proteins, lactacystin increased the level of these proteins. 4. Our data suggest that a reduced level of N-myc and cyclin B1 proteins is critical to commence differentiation, and this can be blocked by a proteasome inhibitor, leading to cell death. Concomitant induction of differentiation and proteasome inhibition, may, therefore, be potentially useful for the treatment of human neuroblastomas.

Topics: Acetylcysteine; Animals; Cell Differentiation; Cyclic AMP; Cyclin B; Cyclin B1; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Flow Cytometry; Gene Expression Regulation, Neoplastic; Genes, myc; Green Fluorescent Proteins; Humans; Leupeptins; Luminescent Proteins; Mice; Multienzyme Complexes; Neuroblastoma; Proteasome Endopeptidase Complex; Retroviridae; Transcription, Genetic; Transfection; Tumor Cells, Cultured

Many cytokines and growth factors activate common signal transduction pathways and yet are able to elicit distinct cell-specific responses. We are defining mechanisms regulating signalling molecules in order to understand how cytokines can produce unique responses. It was found that individual members of the signal transducer and activator of transcription (STAT) family are regulated by ciliary neurotrophic factor (CNTF) and by protein kinase C. Treatment of SH-SY5Y human neuroblastoma cells with the phorbol ester, 12- O -tetradecanoylphorbol 13-acetate (TPA), for 4-5 h caused a 60% decline in both STAT2 and STAT3 levels and no decline in levels of STATs 1, 5 or 6, or in Jaks 1 or 2. The decline in STAT3 was inhibited by treatment with MG132, an inhibitor of proteasome-dependent protein degradation. Treatment of cells with CNTF induced a rapid tyrosine phosphorylation of STAT3 followed by a time-dependent decay of this signal. Loss of tyrosine phosphorylated STAT3 was inhibited by MG132 but did not require protein kinase C activity. These results suggest that STAT3 availability can be controlled by proteasome-dependent pathways activated either by protein kinase C or by cytokines.

Topics: Ciliary Neurotrophic Factor; Cysteine Endopeptidases; Cysteine Proteinase Inhibitors; Cytokines; DNA-Binding Proteins; Humans; Leupeptins; Multienzyme Complexes; Nerve Tissue Proteins; Neuroblastoma; Proteasome Endopeptidase Complex; Protein Kinase C; STAT2 Transcription Factor; STAT3 Transcription Factor; Tetradecanoylphorbol Acetate; Trans-Activators; Tumor Cells, Cultured

Extensive necrotic death of MSN neuroblastoma cells could be induced after incubation with the calcium ionophore, A23187. The reaction was concentration-dependent and time course-dependent. Levels of the 66 kd/alpha-internexin neurofilament protein (NF-66) and the cognate heat shock protein 70 (Hsc 70) decreased during the Ca2+-activated cell death. Addition of the calcium chelator, ethylene glycol-bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid (EGTA) restored the normal level of NF-66 and partially that of the Hsc 70. Use of either calpain I or calpain II inhibitor could alleviate the reduction of 66 kd protein during the ionophore treatment whereas only calpain I inhibitor treatment was effective in restoring the normal level of the Hsc 70. Neither of these calpain inhibitors could block the ionophore triggered cell death. EGTA was toxic to cells in a wide range of concentration suggesting a calcium-independent activation of cell death mechanism.

Topics: Calcimycin; Calpain; Carrier Proteins; Cell Death; HSC70 Heat-Shock Proteins; HSP70 Heat-Shock Proteins; Humans; Intermediate Filament Proteins; Leupeptins; Nerve Tissue Proteins; Neuroblastoma; Neurofibrils; Oligopeptides; Tumor Cells, Cultured

We have previously shown that the degradation of c-myc and N-myc in vitro is mediated by the ubiquitin system. However, the role of the system in targeting the myc proteins in vivo and the identity of the conjugating enzymes and possible ancillary proteins involved has remained obscure. Here we report that the degradation of the myc proteins in cells is inhibited by lactacystin and MG132, two inhibitors of the 20S proteasome. Inhibition is accompanied by accumulation of myc-ubiquitin conjugates. Dissection of the ancillary proteins involved revealed that the high-risk human papillomavirus oncoprotein E6-16 stimulates conjugation and subsequent degradation of the myc proteins in vitro. Expression of E6-16 in cells results in significant shortening of the t1/2 of the myc proteins with subsequent decrease in their cellular level. Analysis of the conjugating enzymes revealed that under basal conditions the proteins can be conjugated by two pairs of E2s and E3s-E2-14 kDa and E3alpha involved in the "N-end rule" pathway, and E2-F1 (UbcH7) and E3-Fos involved also in conjugation of c-Fos. In the presence of E6-16, a third pair, E2-F1 and E6-AP mediate conjugation of myc by means of a mechanism that appears to be similar to that involved in the targeting of p53, formation of a myc. E6.E6-AP targeting complex. It is possible that in certain cells E6-mediated targeting of myc prevents myc-induced apoptosis and thus ensures maintenance of viral infection.

Topics: Acetylcysteine; Cysteine Proteinase Inhibitors; DNA-Binding Proteins; Humans; Leupeptins; Neuroblastoma; Oncogene Proteins, Viral; Papillomaviridae; Papillomavirus Infections; Proto-Oncogene Proteins c-myc; Signal Transduction; Tumor Cells, Cultured; Tumor Virus Infections; Ubiquitins

The turnover of voltage-operated calcium channels was studied in two different human neuroblastoma cell lines (IMR32 and SH-SY5Y) using omega-conotoxin. The 125I-omega-conotoxin bound to surface channels was internalized and degraded by the cells in a time- and temperature-dependent manner. The radioactive degradation products released in the medium were all trichloroacetic acid soluble and no longer recognized by anti-omega-conotoxin antibodies. Altering the pH of intracellular organelles with chloroquine and inhibiting lysosomal proteases with leupeptin reduced 125I-omega-conotoxin degradation but had no effect on its internalization. Postlabeling measurements showed that the rates of 125I-omega-conotoxin internalization and degradation were equal to the rate of channel removal from the cell surface after protein synthesis inhibition. The rate of removal of omega-conotoxin binding sites was parallel to the rate of loss of functional channels, as measured by means of the fura-2 technique. Drug-induced differentiation of human neuroblastoma cells slowed down channel internalization and degradation rates, leading to the known increased expression of plasma membrane calcium channels in differentiated cells. On the other hand, both human (from Lambert-Eaton myasthenic patients) and murine (from immunized mice) anti-channel antibodies increased the rates of channel internalization and degradation, leading to channel downregulation. The activity of presynaptic calcium channels is already known to be acutely modulated by a number of different agents (e.g., hormones and neurotransmitters); our studies suggest that a different form of channel modulation (changes in the number of channels due to interference with channel turnover) may be active over a longer time scale in neurons.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Antibodies; Calcium Channels; Cell Differentiation; Chloroquine; Electrophysiology; Humans; Leupeptins; Neuroblastoma; omega-Conotoxins; Peptides, Cyclic; Temperature

Mouse NB2a/dl neuroblastoma cells elaborate axonal neurites in response to various chemical treatments including dibutyryl cyclic AMP and serum deprivation. Hirudin, a specific inhibitor of thrombin, initiated neurite outgrowth in NB2a/dl cells cultured in the presence of serum; however, these neurites typically retracted within 24 h. The cysteine protease inhibitors leupeptin and N-acetyl-leucyl-leucyl-norleucinal (CI; preferential inhibitor of micromolar calpain but also inhibits millimolar calpain) at 10(-6) M considerably enhanced neurite outgrowth induced by serum deprivation, but could not induce neuritogenesis in the presence of serum. A third cysteine protease inhibitor, N-acetyl-leucyl-leucyl-methional (CII; preferential inhibitor of millimolar calpain but also inhibits micromolar calpain), had no detectable effects by itself. Cells treated simultaneously with hirudin and either leupeptin, CI, or CII elaborated stable neurites in the presence of serum. Cell-free enzyme assays demonstrated that hirudin inhibited thrombin but not calpain, CI and CII inhibited calpain but not thrombin, and leupeptin inhibited both proteases. These results imply that distinct proteolytic events, possibly involving more than one protease, regulate the initiation and subsequent elongation and stabilization of axonal neurites. Since the addition of exogenous thrombin or calpain to serum-free medium did not modify neurite outgrowth, the proteolytic events affected by these inhibitors may be intracellular or involve proteases distinct from thrombin or calpain.

Topics: Animals; Axons; Blood; Calpain; Endopeptidases; Hirudins; Leupeptins; Neuroblastoma; Osmolar Concentration; Protease Inhibitors; Thrombin; Tumor Cells, Cultured

Morphological differentiation of neuroblastoma cells (NB15) was induced by cAMP effectors in the presence and absence of serine protease inhibitors. In all conditions tested, the percent differentiation was inhibited by protease inhibitors antipain, diisopropylfluorophosphate (DFP), leupeptin, and soybean trypsin inhibitor (SBTI). The level of morphological differentiation obtained in medium containing fetal calf serum was significantly less than the percent differentiation obtained with serum-free medium alone, so serum-free medium was the principal method of induction and comparisons were made to control uninduced cultures or cultures induced with the phosphodiesterase inhibitor R020-1724. Secreted or cell surface caseinolytic protease activity was higher in differentiating cells than in control cultures and was inhibited by the serine protease inhibitors. The effects of the protease inhibitors on growth and differentiation are discussed.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; Adenylyl Cyclases; Animals; Antipain; Cell Differentiation; Cyclic AMP; Isoflurophate; Leupeptins; Mice; Neuroblastoma; Trypsin Inhibitor, Kunitz Soybean

Topics: Animals; Carrier Proteins; Cell Line; Cell Membrane; Hippocampus; Imipramine; In Vitro Techniques; Kinetics; Leupeptins; Neuroblastoma; Phospholipases A; Pronase; Rats; Receptors, Drug; Temperature