Search

T-2 toxin is the most acutely toxic trichothecene mycotoxin: it inhibits protein, DNA and RNA synthesis. The main goal of this study was to evaluate the rate of DNA damage caused by T-2 toxin in porcine mononuclear cells in increasing concentrations (0.1, 0.5 and 1.0 μmol) and after two different incubation periods (24 and 42 h). The lowest concentration caused DNA damage and about 50% of the treated cells could be categorised as having 1 to 4 scores in comet assay. In parallel with the increase of T-2 toxin concentration, the frequency of intact lymphocytes decreased from 50.2% (0.1 μM) to 36.3% (1.0 μM) in the first 24 h. In case of score 3, the highest concentration of T-2 toxin resulted in a 5-fold change, as compared to the lowest dose. Cells with score 4 were found only after exposure to 1.0 μM T-2 toxin. The exposure time did not have a significant effect on the results, while concentration did (P < 0.0001). However, a significant interaction between concentration and time as fixed factors (P < 0.0001) was found. When these were combined as a single factor, the results showed a significant toxin treatment effect on the results. It was concluded that a time- and dose-dependent DNA damaging effect of T-2 toxin could be demonstrated using peripheral blood mononuclear cells from healthy pigs by comet assay.

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.

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.

Invasiveness is a common feature of trophoblast and tumors; however, while tumor invasion is uncontrolled, trophoblast invasion is strictly regulated. Both trophoblast and tumor cells express high levels of the immunomodulatory progesterone-induced blocking factor (PIBF), therefore, we aimed to test the possibility that PIBF might be involved in invasion. To this aim, we used PIBF-silenced or PIBF-treated trophoblast (HTR8/Svneo, and primary trophoblast) and tumor (HT-1080, A549, HCT116, PC3) cell lines. Silencing of PIBF increased invasiveness as well as MMP-2,-9 secretion of HTR8/SVneo, and decreased those of HT-1080 cells. PIBF induced immediate STAT6 activation in both cell lines. Silencing of IL-4Rα abrogated all the above effects of PIBF, suggesting that invasion-related signaling by PIBF is initiated through the IL-4Rα/PIBF-receptor complex. In HTR-8/SVneo, PIBF induced fast, but transient Akt and ERK phosphorylation, whereas in tumor cells, PIBF triggered sustained Akt, ERK, and late STAT3 activation. The late signaling events might be due to indirect action of PIBF. PIBF induced the expression of EGF and HB-EGF in HT-1080 cells. The STAT3-activating effect of PIBF was reduced in HB-EGF-deficient HT-1080 cells, suggesting that PIBF-induced HB-EGF contributes to late STAT3 activation. PIBF binds to the promoters of IL-6, EGF, and HB-EGF; however, the protein profile of the protein/DNA complex is different in the two cell lines. We conclude that in tumor cells, PIBF induces proteins, which activate invasion signaling, while—based on our previous data—PIBF might control trophoblast invasion by suppressing proinvasive genes. Hungarian National Research Fund (OTKA 77717), from theUniversity of Pecs (34039/KA-PostDoc12-03) and by TÁMOP-4.2.1/B-10/1-2010-0002. Deposited by bulk import

Problem: The PIBF1-gene contains 18 exons and encodes a protein of 757 aminoacid residues with an 89-kDa molecular mass. We aimed to identify the receptor-binding region of the molecule, by its ability to activate signal transduction via JAK1/STAT6-pathway in peripheral lymphocytes. Materials and Methods: Recombinant PIBF constructs and small synthetic PIBF analogues representing different regions of the molecule were designed. EMSA was performed to detect nuclear translocation of STAT6-dimers while intracellular STAT6-phosphorylation was tested by Western blotting. PIBF-receptor binding ability of the selected analogue was confirmed by confocal microscopy. Results: Nuclear translocation of STAT6-dimers was induced by PN1(exons 2-3-4), PN3(exons 8-9), PN5(exons 3-14-15-16) constructs. Peptide-5 (in PN1) and 8 (in PN3-4) were capable to activate STAT6-phosphorylation. BodipyFL- labelled peptide-8 bound to the PIBF-receptor and was internalized in a time-dependent manner. Conclusions: These data suggest that the receptor-binding region of PIBF might be a conformational structure that includes remote sequences.

Glucocorticoids (GC) are important in the regulation of selection and apoptosis of CD4+CD8+ double-positive (DP) thymocytes. The pronounced GC-sensitivity of DP thymocytes, observed earlier, might be due to the combination of classical (genomic) and alternative (non-genomic) glucocorticoid receptor (GR) signaling events modifying activation or apoptotic pathways. In particular, the previously demonstrated mitochondrial translocation of activated GR in DP thymocytes offered a fascinating explanation for their pronounced GC-induced apoptosis sensitivity. However, the fine molecular details how the mitochondrial translocation of GR might regulate apoptosis remained unclear. Therefore, in the present study, we intended to examine which apoptotic pathways could be involved in GC-induced thymocyte apoptosis. Furthermore we investigated the potential relationship between the GR and Bcl-2 proteins. Using an in vitro test system, thymocytes from 4-week-old BALB/c mice, were treated with the GC-analogue dexamethasone (DX). Bax accumulated in mitochondria upon DX treatment. Mitochondrial GR showed association with members of the Bcl-2 family: Bak, Bim, Bcl-xL. Elevated Cytochrome C, and active caspase-3, -8, and -9 levels were detected in thymocytes after DX treatment. These results support the hypothesis that in early phases of GC-induced thymocyte apoptosis, the mitochondrial pathway plays a crucial role, confirmed by the release of Cytochrome C and the activation of caspase-9. The activation of caspase-8 was presumably due to cross-talk between apoptotic signaling pathways. We propose that the GC-induced mitochondrial accumulation of Bax and the interaction between the GR and Bim, Bcl-xL and Bak could play a role in the regulation of thymocyte apoptosis.