Prolonged treatment with the proteasome inhibitor MG-132 induces apoptosis in PC12 rat pheochromocytoma cells.

Rat pheochromocytoma (PC12) cells were treated with the proteasome inhibitor MG-132 and morphological changes were recorded. Initially, neuronal differentiation was induced but after 24 h signs of morphological deterioration became apparent. We performed nuclear staining, flow cytometry and WST-1 assay then analyzed signal transduction pathways involving Akt, p38 MAPK (Mitogen-Activated Protein Kinase), JNK (c-Jun N-terminal Kinase), c-Jun and caspase-3. Stress signaling via p38, JNK and c-Jun was active even after 24 h of MG-132 treatment, while the survival-mediating Akt phosphorylation declined and the executor of apoptosis (caspase-3) was activated by that time and apoptosis was also observable. We examined subcellular localization of stress signaling components, applied kinase inhibitors and dominant negative H-Ras mutant-expressing PC12 cells in order to decipher connections of stress-mediating pathways. Our results are suggestive of that treatment with the proteasome inhibitor MG-132 has a biphasic nature in PC12 cells. Initially, it induces neuronal differentiation but prolonged treatments lead to apoptosis.

Partial p53-dependence of anisomycin-induced apoptosis in PC12 cells.

The bacterial antibiotic anisomycin is known to induce apoptosis by activating several mitogen-activated protein kinases and by inhibiting protein synthesis. In this study, the influence of p53 protein on the apoptosis-inducing effect of anisomycin was investigated. The effect of protein synthesis-inhibiting concentration of anisomycin on apoptotic events was analyzed using Western blot, DNA fragmentation, and cell viability assays in wild-type PC12 and in mutant p53 protein expressing p143p53PC12 cells. Anisomycin stimulated the main apoptotic pathways in both cell lines, but p143p53PC12 cells showed lower sensitivity to the drug than their wild-type counterparts. Anisomycin caused the activation of the main stress kinases, phosphorylation of the p53 protein and the eukaryotic initiation factor eIF2α, proteolytic cleavage of protein kinase R, Bid, caspase-9 and -3. Furthermore, anisomycin treatment led to the activation of TRAIL and caspase-8, two proteins involved in the extrinsic apoptotic pathway. All these changes were stronger and more sustained in wtPC12 cells. In the presence of the dominant inhibitory p53 protein, p53- dependent genes involved in the regulation of apoptosis may be less transcribed and this can lead to the decrease of apoptotic processes in p143p53PC12 cells.

Partial Protection of PC12 Cells from Cellular Stress by Low-Dose Sodium Nitroprusside Pre-treatment.

The PC12 rat pheochromocytoma cell line is an in vitro model system widely used for the investigation of intracellular signaling events contributing to neuronal differentiation and cell death. We found earlier that the nitric oxide donor compound sodium nitroprusside (SNP) induced apoptosis of PC12 cells if it was applied in high concentration (400 µM). Yoshioka et al. (J Pharmacol Sci 101:126-134, 2006) reported that cell death evoked by cytotoxic concentrations of SNP could be prevented by a 100 µM SNP pre-treatment in a murine macrophage cell line. The apoptosis caused by toxic-dose SNP treatment (400 µM) could be partially overcome in PC12 cells as well by the low-dose SNP pre-treatment. The partial inhibition of apoptosis was accompanied by reduced phosphorylation of certain proteins (such as stress-activated protein kinases, the p53, and the eIF2α proteins), decreased caspase activation, and less intense internucleosomal DNA fragmentation. The 100 µM SNP pre-treatment reduced the pro-apoptotic potential of certain other stress stimuli (serum withdrawal, cisplatin and tunicamycin treatments) as well, although the underlying biochemical changes were not entirely uniform. On the contrary, the 100 µM SNP pre-treatment was unable to prevent cell death caused by the protein synthesis inhibitor anisomycin. Further clarification of the above-mentioned processes may be important in understanding the mechanisms by which mild nitrosative stress protects cells against certain forms of cellular stress conditions.

Sensitivity of Human Malignant Melanoma Cell Lines to Newcastle Disease Virus.

BACKGROUND/AIM: Virotherapy may be a promising alternative to chemotherapy of malignant melanoma. In clinical trials using strains of Newcastle disease virus (NDV), only a fraction of patients with cancer responded to virotherapy. In the present study, we tried to find a correlation between the susceptibility of human melanoma cell lines to NDV and growth factor signaling pathways. MATERIALS AND METHODS: Using an ATP assay, cytotoxicity of an NDV strain (MTH-68/H) was tested in 13 human melanoma cell lines. The activation state of growth factor signaling pathways was studied by the analysis of key signaling proteins. RESULTS: MTH-68/H was found to be cytotoxic in all melanoma cells tested, but the IC50 values varied significantly. No correlation between the IC50 values and the rate of extracellular signal-regulated kinase (ERK) and AKT phosphorylation and phosphatase and tensin homologue (PTEN) expression was found. CONCLUSION: Susceptibility of tumor cells to NDV may be affected by alterations other than those of RAS/ERK and phosphatidylinositol 3-kinase (PI3K)/AKT signaling in uninfected cells.

Problem-solving test: Southwestern blotting.

Terms to be familiar with before you start to solve the test: Southern blotting, Western blotting, restriction endonucleases, agarose gel electrophoresis, nitrocellulose filter, molecular hybridization, polyacrylamide gel electrophoresis, proto-oncogene, c-abl, Src-homology domains, tyrosine protein kinase, nuclear localization signal, cDNA, deletion mutants, expression plasmid, transfection, RNA polymerase II, promoter, Shine-Dalgarno sequence, polyadenylation element, affinity chromatography, Northern blotting, immunoprecipitation, sodium dodecylsulfate, autoradiography, tandem repeats.

The role of the p53 protein in nitrosative stress-induced apoptosis of PC12 rat pheochromocytoma cells.

PC12 rat pheochromocytoma cells are widely used to investigate signaling pathways. The p143p53PC12 cell line expresses a Val143Ala mutant p53 protein that is less capable of binding to the p53 consensus site in DNA than its wild-type counterpart. Nitric oxide (NO), depending on its concentration, is able to activate several signal transduction pathways. We used sodium nitroprusside (SNP), an NO donor compound, to analyze NO-induced cellular stress in order to clarify the mechanism and role of nitrosative stress in pathological processes, including inflammation and cancer. SNP caused cell death when applied at a concentration of 400 µM, p143p53PC12 cells showing higher sensitivity than wild-type PC12 cells. The mechanisms leading to the increased SNP-sensitivity of p143p53PC12 cells were then investigated. The 400-µM SNP treatment caused stress kinase activation, phosphorylation of the eukaryotic initiation factor eIF2α and p53 protein, proteolytic activation of protein kinase R, caspase-9, and caspase-3, p53 stabilization, CHOP induction, cytochrome c release from mitochondria, and a decline in the level of the Bcl-2 protein in both cell lines. All these SNP-induced changes were more robust and/or permanent in cells with the mutant p53 protein. We thus conclude that (1) the main cause of the SNP-induced apoptosis of PC12 cells is the repression of the bcl-2 gene, evoked through p53 stabilization, stress kinase activation, and CHOP induction; (2) the higher SNP sensitivity of p143p53PC12 cells is the consequence of the stronger and earlier activation of the intrinsic apoptotic pathway.

Overexpression of CREB protein protects from tunicamycin-induced apoptosis in various rat cell types.

Endoplasmic reticulum (ER) stress plays an essential role in unfolded protein response induced apoptosis contributing to several pathological conditions. Glycogen synthase kinase-3ß (GSK-3ß) plays a central role in several apoptotic signaling, including ER stress, as the active form of GSK-3ß induces apoptosis. The phosphorylation of cAMP responsive element (CRE) binding protein (CREB) Ser-133 (S133) residue is the end-point of various signaling pathways, like growth factor signaling, while the Ser-129 (S129) residue is phosphorylated by GSK-3ß. The significance of the ubiquitously expressed transcription factor CREB is demonstrated in prolonged, tunicamycin (TM)-induced ER stress in this study. In the experiments wild-type (wt) CREB, S129Ala, S133Ala or S129Ala-S133Ala mutant CREB expressing PC12 rat pheochromocytoma cell lines showed increased survival under TM-evoked prolonged ER stress compared to wtPC12 cells. After TM treatment ER stress was activated in all PC12 cell types. Lithium and SB-216763, the selective, well-known inhibitors of GSK-3ß, decreased TM-induced apoptosis and promoted cell survival. The proapoptotic BH3-only Bcl-2 family member Bcl-2-interacting mediator of cell death (Bim) level was decreased in the different CREB overexpressing PC12 cells as a result of TM treatment. CREB overexpression also inhibited the sequestration of Bim protein from tubulin molecules, as it was demonstrated in wtPC12 cells. Transient expression of wtCREB diminished TM-induced apoptosis in wtPC12, Rat-1 and primary rat vascular smooth muscle cells. These findings demonstrate a novel role of CREB in different cell types as a potent protector against ER stress.

Gene expression profiling in PC12 cells infected with an oncolytic Newcastle disease virus strain.

Although the oncolytic potential of natural, non-engineered Newcastle disease virus (NDV) isolates are well-known, cellular mechanisms determining NDV sensitivity of tumor cells are poorly understood. The aim of the present study was to look for gene expression changes in PC12 pheochromocytoma cells infected with an attenuated NDV strain that may be related to NDV susceptibility. PC12 cells were infected with the NDV strain MTH-68/H for 12h at a titer corresponding to the IC50 value. Total cytoplasmic RNA samples isolated from control and MTH-68/H-infected cells were analyzed using a rat specific Affymetrix exon chip. Genes with at least 2-fold increase or decrease in their expression were identified. MTH-68/H-induced gene expression changes of 9 genes were validated using quantitative reverse transcriptase PCR. A total of 729 genes were up- and 612 genes were down-regulated in PC12 cells infected with MTH-68/H. Using the DAVID functional annotation clustering tool, the up- and down-regulated genes can be categorized into 176 and 146 overlapping functional gene clusters, respectively. Gene expression changes affecting the most important signaling mechanisms (Toll-like receptor signaling, RIG-I-like receptor signaling, interferon signaling, interferon effector pathways, apoptosis pathways, endoplasmic reticulum stress pathways, cell cycle regulation) are analyzed and discussed in detail in this paper. NDV-induced gene expression changes described in this paper affect several regulatory mechanisms and dozens of putative key proteins that may determine the NDV susceptibility of various tumors. Further characterization of these proteins may identify susceptibility markers to predict the chances of virotherapeutic treatment of human tumors.

Problem-solving test: E2F: a family of S phase regulating transcription factors.

Terms to be familiar with before you start to solve the test: transcription factors, gene induction, DNA synthesis, S phase, retinoblastoma, immunoprecipitation, radioactive labeling, oligonucleotide, enhancer element, polyacrylamide gel electrophoresis, autoradiography, gel retardation assay

Problem-solving test: conditional gene targeting using the Cre/loxP recombination system.

Terms to be familiar with before you start to solve the test: gene targeting, knock-out mutation, bacteriophage, complementary base-pairing, homologous recombination, deletion, transgenic organisms, promoter, polyadenylation element, transgene, DNA replication, RNA polymerase, Shine-Dalgarno sequence, restriction endonuclease, polymerase chain reaction, primer, transcription, SV40 virus, malignant transformation, spacer region, gene expression, agarose gel electrophoresis, ethidium bromide staining, Southern-blotting, probe, reporter gene.

The role of Src protein in the process formation of PC12 cells induced by the proteasome inhibitor MG-132.

The PC12 (rat pheochromocytoma) cell line is a popular model system to study neuronal differentiation. Upon prolonged nerve growth factor (NGF) exposure these tumor cells stop to divide, become polygonal, grow projections and start to look and behave like sympathetic neurons. Differentiation of PC12 cells can also be induced by peptidyl-aldehyde proteasome inhibitors, such as Z-Leu-Leu-Leu-al (also known as MG-132) or via infection of the cells with Rous sarcoma virus. The signal transduction pathways underlying process formation, however, are still not fully understood. The liganded NGF receptor initiates a protein kinase cascade a member of which is Extracellular Signal-Regulated Kinase (ERK). Active ERK1/2 enzymes phosphorylate various cytoplasmic proteins and can also be translocated into the nucleus, where they regulate gene expression by activating key transcription factors. Using immunological methods we detected phosphorylation of TrkA, prolongedactivation of Src, and ERK1/2 with nuclear translocation of the latter during MG-132-induced process formation of PC12 cells. Activated Src remained predominantly cytoplasmic. MG-132-induced sustained ERK1/2 activation, nuclear translocation and neuritogenesis required the intact function of Src since these phenomena were markedly reduced or failed upon chemical inhibition of Src tyrosine protein kinase activity.

The effect of sodium nitroprusside on survival and stress signaling in PC12 rat phaeochromocytoma cells expressing a dominant negative RasH mutant protein.

Toxic concentrations of the second messenger nitric oxide cause cellular stress leading to cell death. Ras proteins, possible targets of nitric oxide-induced nitrosylation, may act as mediators in nitrosative stress. To analyze the possible involvement of Ras proteins in nitric oxide cytotoxicity, a PC12 rat phaeochromocytoma cell line expressing a dominant negative Ras mutant protein was used in this study. Cytotoxic concentrations of the nitric oxide donor sodium nitroprusside activated several proapoptotic mechanisms, including stimulation of the stress kinase pathways mediated by c-Jun N-terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK), inhibition of the translation initiation factor eIF2α, induction and phosphorylation of the p53 protein, and inhibited Akt-mediated antiapoptotic signaling, independent of Ras function. Simultaneously, Ras-dependent stimulation of the prosurvival ERK pathway was also observed, followed by an increased activation of the caspase-9/caspase-3 cascade in cells with impaired Ras function. It is concluded that nitric oxide stimulation of multiple signaling pathways contributes to the cell death program, whereas concomitant activation of the Ras/ERK pathway provides a certain degree of protection.

Partial rescue of geldanamycin-induced TrkA depletion by a proteasome inhibitor in PC12 cells.

In this work we tried to identify mechanisms that could explain how chemical inhibition of heat-shock protein 90 reduces nerve growth factor signaling in rat pheochromocytoma PC12 cells. Geldanamycin is an antibiotic originally discovered based on its ability to bind heat-shock protein 90. This interaction can lead to the disruption of heat-shock protein 90-containing multimolecular complexes. It can also induce the inhibition or even degradation of partner proteins dissociated from the 90 kDa chaperone and, eventually, can cause apoptosis, for instance, in PC12 cells. Before the onset of initial apoptotic events, however, a marked decrease in the activity of extracellular signal-regulated kinases ERK 1/2 and protein kinase B/Akt can be observed together with reduced expression of the high affinity nerve growth factor receptor, tropomyosine-related kinase, TrkA, in this cell type. The proteasome inhibitor MG-132 can effectively counteract the geldanamycin-induced reduction of TrkA expression and it can render TrkA and ERK1/2 phosphorylation but not that of protein kinase B/Akt by nerve growth factor again inducible. We have found altered intracellular distribution of TrkA in geldanamycin-treated and proteasome-inhibited PC12 cells that may, at least from the viewpoint of protein localization explain why nerve growth factor remains without effect on protein kinase B/Akt. The lack of protein kinase B/Akt stimulation by nerve growth factor in turn reveals why nerve growth factor treatment cannot save PC12 cells from geldanamycin-induced programmed cell death. Our observations can help to better understand the mechanism of action of geldanamycin, a compound with strong human therapeutical potential.

Problem-solving test: analysis of DNA damage recognizing proteins in yeast and human cells.

Terms to be familiar with before you start to solve the test: DNA repair, germline mutation, somatic mutation, inherited disease, cancer, restriction endonuclease, radioactive labeling, [α-(32) P]ATP, [γ-(32) P]ATP, DNA ligase, polynucleotide kinase, DNA polymerase, terminal transferase, DNA helicase, UV-irradiation, pyrimidine dimer, base modifications, mismatch, polyacrylamide gel electrophoresis, autoradiography, molecular hybridization, satellite DNA, nucleosomes.

Problem-solving test: signal transduction in Philadelphia chromosome positive leukemia cells.

Terms to be familiar with before you start to solve the test: reciprocal translocation, proto-oncogene, gene expression, c-Abl, tyrosine protein kinases, transmembrane protein, G protein, Src protein, malignant transformation, transfection, immunoprecipitation, pre-immune serum, [γ-(32)P]ATP, cAMP, SDS-polyacrylamide gel electrophoresis, autoradiography, Western blot (immunoblot), co-immunoprecipitation, expression vector, cDNA, transient transfection, reporter gene, reporter plasmid, promoter, Ras protein, transformed foci, extracellular signal-regulated kinase.

Problem-solving test: the role of ubiquitination in epidermal growth factor receptor trafficking.

Terms to be familiar with before you start to solve the test: growth factor signaling, epidermal growth factor, tyrosine protein kinase, tyrosine phosphorylation, ubiquitin, monoubiquitination, polyubiquitination, site-directed mutagenesis, transfection, expression vector, cDNA, immunoprecipitation, SDS-polyacrylamide gel electrophoresis, Western blotting, radioactive labeling, receptor-mediated endocytosis.

Sodium nitroprusside, a nitric oxide donor, fails to bypass the block of neuronal differentiation in PC12 cells imposed by a dominant negative Ras protein.

Nitric oxide (NO) is a mediator of a diverse array of inter- and intracellular signal transduction processes. The aim of the present study was to analyze its possible role as a second messenger in the process of neuronal differentiation of PC12 pheochromocytoma cells. Upon NGF treatment wildtype PC12 cells stop dividing and develop neurites. In contrast, a PC12 subclone (designated M-M17-26) expressing a dominant-negative mutant Ras protein keeps proliferating and fails to grow neurites after NGF treatment. Sodium nitroprusside (SNP), an NO donor, was found to induce the p53 protein and to inhibit proliferation of both PC12 and M-M17-26 cells, but failed to induce neuronal differentiation in these cell lines. Key signaling pathways (the ERK and Akt pathways) were also not affected by SNP treatment, and the phosphorylation of CREB transcription factor was only slightly stimulated. It is thus concluded from the results presented in this paper that NO is unable to activate signaling proteins acting downstream or independent of Ras that are required for neuronal differentiation.

Analysis of three properties of Newcastle disease virus for fighting cancer: tumor-selective replication, antitumor cytotoxicity, and immunostimulation.

Newcastle disease virus (NDV), a bird paramyxovirus, is an antitumor agent which has shown benefits to cancer patients. Its antineoplastic efficacy appears to be associated with three properties of the virus: 1. Selective replication in tumor cells. This feature can be studied at the RNA level, for example by RT-PCR, and at the protein level by immunochemistry. 2. Oncolytic properties (of some strains). The use of cultures of tumor cell lines represents a selective model to study direct viral oncolysis at the cellular level. The capacity of NDV to lyse tumor cells can be analyzed in vitro using cytotoxic assays based on the WST1 chemical reagent. The endoplasmic reticulum stress, which is induced by infection with the oncolytic NDV strain MTH-68/H and which plays an important role in the viral oncolytic effects, can be analyzed by Western blotting using specific monoclonal antibodies. Such stress appears as a key component of NDV cytotoxicity. 3. Immunostimulatory capacity. We describe an in vitro test called "Tumor Neutralisation Assay" which allows the analysis of bystander antitumor immune effects induced in human peripheral blood mononuclear cells by NDV. There are two variants, one for oncolytic NDV strains and the other one for nonlytic NDV strains. NDV may use several mechanisms to exert its tumor-killing action: direct cytotoxicity against cancer cells but also nonspecific as well as active-specific antitumor immune responses from the host organism. All the methods described here allow to evaluate the different oncolytic and immunostimulatory capacities of various strains of NDV. They are crucial to harness optimal antitumor activity by appropriate combinations of virus strains and application regimens.

A simple fluorescent labeling technique to study virus adsorption in Newcastle disease virus infected cells.

The present study demonstrates that the fluorescent general membrane dyes PKH67 and PKH26 are suitable to label Newcastle disease virus, an enveloped virus belonging to the family of paramyxoviridae. Adsorption of the labeled virus particles was tracked, visualized and quantitated using confocal laser scanning microscopy. The specificity of PKH-labeling was determined by colocalization analysis of the PKH signal with NDV-specific immunolabeling, and by using mock-infected controls and infection with detergent-pretreated labeled virus particles. The infectivity of the NDV particles was not affected by the labeling procedure as indicated by the results of a cytotoxicity ATP assay, an apoptosis assay and detection of virus-specific RNA and protein by qPCR and Western blotting, respectively, in cells infected with PKH-labeled and unlabeled virus particles. This technique can be used as an inexpensive, sensitive and rapid alternative method in the analysis of adsorption and internalization of enveloped viruses by the infected cells.