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Szeberenyi J.,University of Pecs | Szeberenyi J.,Signal Transduction Research Group
Biochemistry and Cell Biology

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. © 2013 Published by NRC Research Press. Source

Varga J.,University of Pecs | Varga J.,Signal Transduction Research Group | Bator J.,University of Pecs | Bator J.,Signal Transduction Research Group | And 9 more authors.
Cell and Tissue Research

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. © 2014, Springer-Verlag Berlin Heidelberg. Source

Pap M.,University of Pecs | Pap M.,Signal Transduction Research Group | Bator J.,University of Pecs | Bator J.,Signal Transduction Research Group | And 2 more authors.
Anticancer Research

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. Source

Varga J.,University of Pecs | Varga J.,Signal Transduction Research Group | Bator J.,University of Pecs | Bator J.,Signal Transduction Research Group | And 7 more authors.
Cellular and Molecular Neurobiology

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. © 2015 Springer Science+Business Media New York Source

Balogh A.,University of Pecs | Balogh A.,Signal Transduction Research Group | Bator J.,University of Pecs | Bator J.,Signal Transduction Research Group | And 11 more authors.
Virus Research

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. © 2014 Elsevier B.V. Source

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